Compounds that bind to P185 and methods of using the same

ABSTRACT

Novel peptides and pharmaceutical compositions comprising the same are disclosed. Conjugated compositions peptides linked to detectable agents and/or cytotoxic agents. are disclosed. Method of detecting tumors that have p185 on tumor cell surfaces are disclosed. Methods of preventing transformation of a normal cell into a tumor cell in an individual at risk of developing a tumor having tumor cells which have p185 on their surfaces are disclosed. Methods of treating an individual who has cancer characterized by tumor cells that have a p185 on their cell surfaces are disclosed.

This application is a divisional of U.S. application Ser. No.10/301,499, filed on Nov. 21, 2002, which claimed priority under 35U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60/331,935,filed Nov. 21, 2001, both incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The invention relates to compounds useful for and methods of treatingindividuals suspected of suffering from tumors and preventing tumors inindividuals suspected of being susceptible to the development of tumorsand for detecting and imaging tumors.

BACKGROUND OF THE INVENTION

Significant amounts of time and money have been spent to betterunderstand cancer and searching for ways to prevent and cure cancer. Theresults of these research efforts have provided a greater understandingof the biological and biochemical events that participate in theformation of tumors.

Malignant cells display a variety of characteristics that distinguishthem from normal cells. Recent studies in the molecular genetics ofcancer indicate that certain genes known as oncogenes may play a role inthe transformation of some cells from their normal condition to acancerous condition. Proto-oncogenes, genes closely related to thesegenes, are found in somatic cells of all eukaryotic species examined andhave been highly conserved in evolution; it is thought thatproto-oncogenes normally play critical roles in cellular growth anddevelopment. Oncogene amplification and chromosomal rearrangementsinvolving oncogenes have been detected in a large number of tumors.Furthermore some tumors have been shown to contain activated oncogeneswhich, in DNA transfection assays, are capable of conferring neoplasticproperties upon non-neoplastic rodent fibroblast cell lines.Collectively these studies suggest that alterations in proto-oncogenestructure and function play a critical role in the development ofneoplasia.

Although most oncogene-encoded proteins reside in the nucleus or thecytoplasm, some oncogenes encode proteins that are present as antigenicsites on the cell surface. For example, the erbB-1, erbB-2, erbB-3,erbB-4, fms and ros oncogene products are transmembrane glycoproteinsthat possess extracellular domains. The sis oncogene product may alsoexist in a membrane associated form on the surface of transformed cells.

Another oncogene which encodes a protein that exposes antigenic sites onthe surface of transformed cells has been identified by transfection ofDNA from ethyl nitrosourea-induced rat neuroblastomas into NIH3T3 cells.This oncogene has been termed neu. The homologous human gene is callederbB-2. The erbB-2 gene has been found to be amplified or overexpressedin some human tumors, particularly those of the breast, suggesting thatthis gene may play an imported role in the etiology of human cancer.

The protein encoded by the erbB-2 oncogene is a 185 kDa transmembraneglycoprotein with tyrosine kinase activity, generally known by the namep185. The erbB-2 gene is closely related to the epidermal growth factor(EGF) receptor gene in structure.

The erbB-2 oncogene and p185 has also been found active in humanadenocarcinomas including breast, lung, salivary gland and kidneyadenocarcinomas, as well as prostate neuroblastoma. In human primarybreast cancers, amplification of the erbB-2 oncogene was found in about30% of all malignant tumors examined. Increased stage of malignancy,characterized by large tumor size and increased number of positive lymphnodes as well as reduced survival time and decreased time to relapse,was directly correlated with an increased level of amplification of theerbB-2 gene. The erbB-2 protooncogene is expressed at low levels innormal human tissues. Further, erbB-2 has been associated with 100% ofthe ductal carcinomas studied in situ, Lodato, R. F., et al. (1990)Modern Pathol. 3(4):449.

Current treatments for individuals suffering from carcinomas expressingamplified levels of erbB-2 include surgery, radiation therapy,chemotherapy, immunotherapy and, usually, combinations of two or more ofsuch therapies. Despite advances made in these fields, the mortalityrate among individuals suffering from cancer remains unacceptable high.Complete tumor eradication and total remission is not always theoutcome.

There remains a need for additional modalities in the anti-tumorapproaches and for additional methods of reducing and/or eliminatingtumors in individuals. There is a need for anti-tumor agents which canbe administered as therapeutics to individuals suffering form tumors,particularly tumors with amplified levels of p185.

While changes in diet and behavior can reduce the likelihood ofdeveloping cancer, it has been found that some individuals have a higherrisk of developing cancer than others. Further, those individuals whohave already developed cancer and who have been effectively treated facea risk of relapse and recurrence.

Advancements in the understanding of genetics and developments intechnology as well as epidemiology allow for the determination ofprobability and risk assessment an individual has for developing cancer.Using family health histories and/or genetic screening, it is possibleto estimate the probability that a particular individual has fordeveloping certain types of cancer. Those individuals that have beenidentified as being predisposed to developing a particular form ofcancer can take only limited prophylacetic steps towards reducing therisk of cancer. There is no currently available method or compositionwhich can chemically intervene with the development of cancer and reducethe probability a high risk individual will develop cancer.

Similarly, those individuals who have already developed cancer and whohave been treated to remove the cancer or are otherwise in remission areparticularly susceptible to relapse and reoccurrence. As part of atreatment regimen, such individuals can be immunized against the cancerthat they have been diagnosed as having had in order to combat arecurrence. Thus, once it is known that an individual has had a type ofcancer and is at risk of a relapse, they can be immunized in order toprepare their immune system to combat any future appearance of thecancer.

There is a need for improved preventative agents for individuals with ahigh risk to develop cancer, and for individuals who have had cancerenter remission or be removed (e.g., resected). In cases where the typeof cancer the individual is at risk to develop is known, such as tumorsassociated with erbB-2, there is a need for specific agents which can beadministered to reduce the probability that a predisposed individualwill develop cancer or that a patient in remission will suffer arelapse.

There is a need for therapeutic compositions useful to treat individualsidentified as having p185-associated tumors. There is also a need todevelop prophylacetic compositions for individuals susceptible todeveloping p185-associated tumors.

SUMMARY OF THE INVENTION

The present invention relates to peptides having the Formula I orFormula II:R₁,⁻R₂ ⁻R₃ ⁻R₄ ^(−R) ₅  (I)wherein:

-   -   R₁, is O-benzyloxy or 1-4 amino acid residues including at least        one of tyrosine or phenylalanine;    -   R₂ is a linking moiety which bonds with R₁, R₃ and R₄ such that        a portion of said peptide is cyclicized;    -   R₃ is 5 amino acids;    -   R₄ is a linking moiety which bonds with R₃, R₅ and R₂ such that        a portion of said peptide is cyclicized;    -   R₅ is 1-13 amino acid residues and at least one of which is        tyrosine or phenylalanine;

wherein: R₁, R₂, R₃, R₄, and R₅ taken together, are 20 amino acids orless;

and R₃ is has the formula:R₃₁—R₃₂—R₃₃—R₃₄—R₃₅;wherein:

-   -   R₃₁ is aspartic acid;    -   R₃₂ is glycine;    -   R₃₃ is phenylalanine, tyrosine, tryptophan, histidine,        D-phenylalanine, D-tyrosine, D-tryptophan, or D-histidine;    -   R₃₄ is tyrosine; and    -   R₃₅ is alanine, glycine, proline, D-alanine, D-glycine, or        D-proline; and the carboxy terminus of R_(S) is either amidated        or hydroxylated.        R₆ ⁻R₇ ⁻R₈ ⁻R₉ ⁻R₁₀  (II)        wherein    -   R₆ is 1-4 amino acid residues including at least one of tyrosine        or phenylalanine;    -   R₇ is cysteine;    -   R₈ is 5-7 amino acids;    -   R₉ is cysteine;    -   R₁₀ is 1-13 amino acid residues and at least one of which is        tyrosine or phenylalanine;

wherein: R₆, R₇, R₈, R₉, and R₁₀ taken together, are 20 amino acids orless; and R₈ has the formulaR₈₁—R₈₂—R₈₃wherein

-   -   R₈₁, is glycine-aspartic acid, proline-aspartic acid, or        aspartic acid;    -   R₈₂ is glycine, proline or proline-proline; and    -   R₈₃ is phenylalanine-tyrosine-alanine;        and the carboxy terminus of R₁₀ is either amidated or        hydroxylated.

The present invention relates to pharmaceutical compositions whichcomprise a peptide of Formula I or Formula II in combination with apharmaceutically acceptable carrier or diluent.

The present invention relates to methods of preventing transformation ofa cell which overexposes p185 but is not fully transformed into atransformed tumor cell in an individual at risk of developing a tumor orhaving tumor cells which have p185 on their surfaces. The methodcomprises the steps of: identifying such an individual; andadministering to the individual peptide of Formula II.

The present invention relates to methods of treating an individual whohas cancer characterized by tumor cells that have a p185 on their cellsurfaces. The methods comprise the steps of identifying such anindividual; and administering to the individual, a therapeuticallyeffective amount of a peptide of Formula I or Formula II.

The present invention relates to conjugated compositions that comprise apeptide linked to a detectable agent and/or a cytotoxic agent, whereinthe peptide has either Formula I or Formula II.

The present invention relates to methods of detecting a tumor that hasp185 on tumor cell surfaces. The methods comprise the step ofadministering, to an individual suspected of having such a tumor orbeing susceptible to such a tumor, a conjugated composition describedabove which comprises a peptide of Formula I or Formula II linked to adetectable agent and detecting the presence of localized conjugatedcomposition within the body of the individual.

The present invention relates to pharmaceutical compositions whichcomprise a conjugated compositions described above which comprises apeptide of Formula I or Formula II linked to a detectable agent and/or acytotoxic agent in combination with a pharmaceutically acceptablecarrier or diluent.

The present invention relates to methods of treating an individual whohas cancer characterized by tumor cells that have a p185 on their cellsurfaces. The methods comprise the steps of identifying such anindividual; and administering to the individual, a therapeuticallyeffective amount of a conjugated composition described above whichcomprises a peptide of Formula I or Formula II linked to a cytotoxicagent.

The present invention relates to co-administration of pharmaceuticalcompositions which comprise a peptide of Formula I or Formula II withother treatments for cancer characterized by tumor cells that have ap185 on their cell surfaces, such as Herceptin or tamoxifen.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Biosensor dose dependence curves for binding of AHNP to theimmobilized HER2 receptor. AHNP was injected at 0.5 μM (a), 1 μM (b), 2μM (c), 4 μM (d), and 8 μM (e) concentrations at a flow rate of 20μL/min. Sensorgrams show binding of AHNP to the immobilized HER2 (first300 s) followed by the peptide dissociation from the receptor surface(last 240 s).

FIG. 2. Biacore analysis of the inhibitory effect of AHNP on rhumAb 4D5binding to immobilized HER2. Sensorgrams show binding of 1 nM rhumAb 4D5to HER2 after preinjection of 0 μM (a), 0.35 μM (b), 1.4 μM (c), 5.6 μM(e), and 89 μM (f) AHNP.

Inset shows correlation between the initial rate of rhumAb 4D5 bindingto HER2 and concentration of preinjected AHNP.

FIGS. 3A, 3B and 3C. Molecular models of AHNP analogues. AHNP (A), 6(B), and 8 (C). Cys residues forming the disulfide bond are shown inyellow. The Met residue in the tail region of AHNP (A) and the Lysresidue replacing Met in 6 (b) are shown in red.

FIGS. 4A and 4B. Low-energy conformers of 7. Molecular models of twolow-energy conformers of 7 with the solvent-exposed (A) and buried (B)orientation of the N-terminal Gly. The Gly residue is colored red.

FIG. 5. Molecular model of 4 superimposed with AHNP. Aromatic sidechains at both ends of the disulfide bond are colored red for AHNP andwhite for 4.

FIGS. 6A and 6B. Structure-function analysis of AHNP analogues. Plotsshow correlation between peptides' activities in MTT assays and (A)their receptor-binding affinities, or (B) dissociation rate constantsobtained in Biacore studies.

FIG. 7. Graph representing blood clearance of ^(99m)Tc-labeled AHNP innude mice.

FIGS. 8A and 8B. Radioactive imaging of ^(99m)Tc-labeled AHNP inxenografted tumor tissue compared with normal tissue after 30 min (FIG.89A) and 90 min. (FIG. 8B).

FIG. 9. Graph representing the enhanced growh inhibition of HER-2overexpressing cells using an AHNP analogue in combination withtamoxifen

DETAILED DESCRIPTION Definitions

As used herein, the terms “neu-associated cancer”, “erbB-2-associatedcancer”, “neu-associated tumors”, “erbB-2-associated tumors”,“p185-mediated tumors” and “p185-associated tumors” are meant to referto tumor cells and neoplasms which express the erbB-2 gene to producep185. Examples of erbB-2-associated cancer include many humanadenocarcinomas. Breast, ovary, lung, pancreas, salivary gland andkidney adenocarcinomas and prostate, and some neuroblastoma have beenfound to be erbB-2-associated cancers.

When a therapeutically effective amount of a compound of the inventionis administered to an individual who has erbB-2-associated cancer, theeffect is that the proliferation rate of tumor cells is slowed down oreliminated. As used herein, the term “compound” is meant to refer to apeptide of Formula I or Formula II, a peptide mimetic or a conjugatedcompound comprising a peptide of Formula I or Formula II which is usefulin the method of detecting, imaging, treating or preventingp185-mediated tumors.

As used herein, the term “therapeutically effective amount” is meant torefer to an amount of a compound which produces a medicinal effectobserved as reduction or reverse in tumorigenic phenotype of tumor cellsin an individual when a therapeutically effective amount of a compoundis administered to an individual who is susceptible to or suffering fromp185-mediated tumors. Therapeutically effective amounts are typicallydetermined by the effect they have compared to the effect observed whena composition which includes no active ingredient is administered to asimilarly situated individual.

As used herein, the term “high risk individual” is meant to refer to anindividual who has had a erbB-2-associated tumor either removed or enterremission and who is therefore susceptible to a relapse or recurrence.As part of a treatment regimen for a high risk individual, theindividual can be prophylactically treated to conduct the recurrence ofthe erbB-2-associated tumors. Thus, once it is known that an individualhas had cancer characterized by tumor cells with p185 on their cellsurfaces, the individual can be treated according to the presentinvention to prevent normal cells from transforming into tumor cells.

As used herein, the term “preventing the development of tumors” is meantto refer to preventing the transformation of cells that are not fullytransformed into tumor cells. Thus, the development of tumors refers tothe transformation event which results in the loss of a more normalphenotype and the acquisition of a transformed phenotype. According tosome aspects of the present invention, compounds may be administered toindividuals who are at risk of developing tumors. The prophylaceticadministration of compounds of the invention to high risk individualsresults in the prevention of the transformation event occurring. Cellshaving the more normal phenotype are not converted to the cells havingtransformed phenotype. The compounds of the invention prevent tumorsbefore they are formed by preventing a cell that are not fullytransformed from becoming a cancer cell.

As used herein, the term “prophylactically effective amount” is meant torefer to an amount of a compound which produces a medicinal effectobserved as the prevention of cells that are not fully transformed frombecoming transformed in an individual when a prophylactically effectiveamount of a compound is administered to an individual who is susceptibleto p185-mediated tumors. Prophylactically effective amounts aretypically determined by the effect they have compared to the effectobserved when a composition which includes no active ingredient isadministered to a similarly situated individual.

As used herein, the terms “conformationally restricted peptides”,“constrained peptides” and “conformationally constrained peptides” areused interchangeably and are meant to refer to peptides which, forexample through intramolecular bonds, are conformationally stable andremain in a sufficiently restricted conformation. The compounds have anaffinity to p185 and, when bound to p185 as cells, a biologically activeeffect on cells that have a p185-mediated transformation phenotype.

As used herein, the terms “aromatic amino acids” and “aromatic aminoacid residues” used interchangeably are meant to refer to phenylalanineand tyrosine.

As used herein, the term “exocyclic amino acid residue” is meant torefer to amino acid residues which are linked to cyclicized peptide butwhich are not within the portion of the peptide that makes up thecircularized structure.

As used herein, the term “exocyclic portions” is meant to refer to anamino acid sequence having one or more amino acid residues which islinked to cyclicized peptide but which are not within the portion of thepeptide that makes up the circularized structure.

As used herein, the term “linking moiety” is meant to refer to amolecular component or functional group which is capable of formingbonds with three amino acids. As used herein, the term “linking aminoacid residue” is meant to refer to an amino acid residue that is alinking moiety.

As used herein, the terms “active sequence” and “active region” are usedinterchangeably and are meant to refer to the amino acid sequence of theportion of a compound of the invention that is directly interacts withp185, wherein the interaction is characterized by an affinity betweenthe active portion and p185.

As used herein, the term “chelating linker” is meant to refer tochemical linkers which are used to conjugate an amino acid residue of apeptide sequence to a detectable or cytotoxic agent. Examples includethe macrocyclic polyaminoacetate DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), and DTPA(diethylenetriamine pentaacetate).

“Tamoxifen” refers to tamoxifen citrate. Tamoxifen has a molecularweight of 563.62, the pKa′ is 8.85, the equilibrium solubility in waterat 37° C. is 0.5 mg/ml and in 0.02 N HCl at 37° C., it is 0.2 mg/ml.

Chemically, tamoxifen is the trans-isomer of a triphenylethylenederivative. The chemical name is (Z)₂-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine 2-hydroxy-1,2,3-propanetricarboxylate(1:1).

Description

The present invention relates to constrained peptides that containexocyclic portions including exocyclic amino acids that are aromaticamino acids as well as an active region which specifically binds top185. U.S. Pat. No. 6,100,377 issued Aug. 8, 2000 and entitled“Constrained Peptides” is incorporated herein by reference in itsentirety.

The present invention is useful to therapeutically treat an individualidentified as suffering from erbB-2-associated tumors in order toreverse the transformed phenotype of the tumor cells. The presentinvention is useful to prophylactically treat an individual who ispredisposed to develop erbB-2-associated tumors or who has haderbB-2-associated tumors and is therefore susceptible to a relapse orrecurrence. The present invention is useful to detectably image tumorswith respect to p185 on their surfaces. The present invention is usefulto detect and quantify p185 on all surfaces. The present invention isalso useful to target cytotoxic agents preferentially to tumor tissue asopposed to normal tissue.

The translation product of the erbB-2 oncogene is p185, a transmembraneglycoprotein having tyrosine kinase activity and a molecular weight ofabout 185,000 daltons as determined by carrying out electrophoresis onthe glycoprotein and comparing its movement with marker proteins ofknown molecular weight. Experiments have shown that p185 formshomodimers with other p185 molecules or heterodimers with epidermalgrowth factor receptor (EGFR) and that these dimers exhibit elevatedtyrosine kinase activity which brings about the transformed phenotype incells having such dimers. It is believed that dimerization of p185 withother membrane bound receptors, such as other p185 molecules or EGFR,results in elevated levels of tyrosine kinase activity and thetransformed phenotype.

According to the present invention, compounds bind to p185 and therebyprevent the dimerization with other membrane bound receptors by downmodulation of their surface receptors. When bound to p185, the compoundsof the invention induce internalization of the receptor which results inelimination or reduction of tyrosine kinase activity. The elimination orreduction of tyrosine kinase activity results in an elimination orreduction in cell proliferation levels and a non-transformed, quiescentphenotype. The compounds of the invention cause down-modulation oferbB-2 cell surface reception. When bound to p185, the compounds of theinvention reverse the transformed state of such cells, resulting indecreasing the rate of transformation in cells showing intactnon-activated tyrosine kinase receptors found in normal cells are notaffected by the compounds of the invention and hence are non-toxic.

The compounds of the invention are therefore useful in the treatment ofindividuals suspected of having from p185-mediated tumors. Whenadministered to individuals who have been thusly identified, thecompounds of the invention bind to p185, thereby causing modulation oferbB-2 receptors. The p185 receptor bound to the compound internalizes,and the internalization of the p185 receptor contributes to the decreasein tyrosine kinase activity of the p185 receptors. When the tyrosinekinase activity in the cell is reduced from the elevated levelsassociated with amplified or overexpressed p185, the cell becomesquiescent and displays a non-transformed phenotype.

The compounds of the invention are also useful in the prevention ofp185-mediated tumor formation and therefore in the method ofprophylactically treating high risk individuals from developingp185-mediated tumors. That is, the prophylacetic administration ofcompounds of the invention results in the prevention of cells thatoverexpress p185 from becoming transformed. The cells in the individualswhich would turn into tumors in an untreated individual never becomefully transformed and never become tumors in individuals treated by themethods of the invention. It has been discovered that the stocasticappearance of tumors which appear following removal of tumors orremission can be prevented by administration of compounds of theinvention. When administered to individuals who have been identified asbeing susceptible to or otherwise at risk of developing tumors, thecompounds of the invention bind to p185, thereby preventing and causethe internalization of the receptor/compound complex. The p185 receptorbound to the compound internalizes and the bound p185 receptor does notcontribute the elevation in tyrosine kinase activity associated withdimerized p185 receptors. The tyrosine kinase activity in the cell neverbecome sufficiently elevated and the cell remains non-transformed.

In some embodiments, the compounds of the invention can be labeled orotherwise made detectable. As a detectable compound that binds to p185,the compounds are useful as imaging agents and reagents in diagnosticprocedures that are used to identify a tumor as being a p185-associatedtumor. Labeled compounds of the invention can be administered toindividuals suspected of suffering from p185-associated tumors. Thelabeled compounds will bind to the high density of p185 on cells andthereby accumulate on p185-associated tumor cells. Using standardimaging techniques, the site of the tumors can be detected.

One aspect of the invention therefore relates to methods of imagingp185-associated tumors. Such methods comprise the steps of administeringa detectable compound of the invention to an individuals who issuffering from or susceptible to erbB-2-associated cancer and detectingthe location of the detectable compound within the body of theindividual.

The compounds bind to p185 that is present on cell surfaces and aretherefore useful as diagnostic/characterizing reagents in diagnostickits. When a tissue sample from an individual is contacted with acompound of the invention, the compound will bind to the p185 present oncells. The level of p185 expression can be quantified. Labeled compoundsof the invention are also useful as in vitro reagents to quantify theamount of p185 present in the cell. Such information indicates whetheror not a tumor is p185 mediated and therefore, whether specifictreatments should be used or avoided. Using standard techniques, samplesbelieved to include tumor cells are obtained and contacted with labeledcompounds of the active region of the invention. After removing anyunbound labeled compounds, the quantity of labeled compound bound to thecell or the quantity of removed as unbound labeled compounds isdetermined. The information directly relates to the amount of p185 thecell expresses and thus can be used to determine whether a cell is overexpressing p185. Overexpression of p185 indicates p185-mediatedtransformation. This information is useful in formulating the prognosisand course of treatment to be imposed on the individual. Kits of theinvention comprise detectable compounds of the invention andinstructions for performing assays of the invention. Optionally, kitsmay also contain one or more of the following: containers which comprisepositive controls, containers which comprise negative controls,photographs of representative examples of positive results andphotographs of representative examples of negative results.

According to some embodiments, the present invention provides peptideshaving Formula IR₁,—R₂—R₃—R₄—R₅  (I)wherein:

-   -   R₂, is O-benzyloxy or 1-4 amino acid residues including at least        one of tyrosine or phenylalanine;    -   R₂ is a linking moiety which bonds with R₁, R₃ and R₄ such that        a portion of said peptide is cyclicized;    -   R₃ is 5 amino acids;    -   R₄ is a linking moiety which bonds with R₃, R₅ and R₂ such that        a portion of said peptide is cyclicized;    -   R₅ is 1-13 amino acid residues and at least one of which is        tyrosine or phenylalanine;

wherein: R₁, R₂, R₃, R₄, and R₅ taken together, are 20 amino acids orless;

and R₃ is has the formula:R₃₁—R₃₂—R₃₃—R₃₄—R₃₅;wherein:

-   -   R₃₁, is aspartic acid; R₃₂ is glycine;    -   R₃₃ is phenylalanine, tyrosine, tryptophan, histidine,        D-phenylalanine, D-tyrosine, D-tryptophan, or D-histidine;    -   R₃₄ is tyrosine; and    -   R₃₅ is alanine, glycine, proline, D-alanine, D-glycine, or        D-proline; and the carboxy terminus of R₅ is either amidated or        hydroxylated.

The primary function of R₁, in compounds of the present invention arisesfrom the presence of at least one amino acid that contains an aromaticgroup: i.e., the presence of tyrosine or phenylalanine. The presence ofthe aromatic amino acid at position R₁, results in an increase affinityof the peptide to p185 and an attendant increase in activity of thecompound. In embodiments where additional amino acid residues arepresent, they can present the aromatic amino acid in a more effectiveposition to further increase the affinity and activity of the compound.Additional amino acids that may be present must not eliminate the effectthat the aromatic amino acid has on affinity or activity. Examples ofamino acid sequences which may be used as R₁ are disclosed in U.S.Serial Number 6,100,377. In some embodiments, the additional amino acidsare present as a site for linkage to detectable labels or moieties. Insome embodiments, the additional amino acids are present as a site fordimerization with other peptides; either for formation of homodimerswith each other or heterodimers with other peptides. In some preferredembodiments, R₁ is 1-4 amino acids. In some preferred embodiments, R₁ is4 amino acids. In some preferred embodiments, R₁ is 3 amino acids. Insome preferred embodiments, R₁ is 2 amino acids. In some preferredembodiments, R, is 1 amino acid. In some preferred embodiments, R₁consists of Phe, dPhe, Tyr, dTyr, Gly-Phe, Gly-dPhe, Gly-Tyr, Gly-dTyr,Ala-Phe, Ala-dPhe, Ala-Tyr, Ala-dTyr, Lys-Phe, Lys-dPhe, Lys-Tyr,Lys-dTyr, Gly-Gly-Phe, Gly-Gly-dPhe, Gly-Gly-Tyr, Gly-Gly-dTyr,Gly-Gly-Gly-Phe (SEQ ID NO: 1), Gly-Gly-Gly-dPhe (SEQ ID NO:2),Gly-Gly-Gly-Tyr (SEQ ID NO:3), Gly-Gly-Gly-dTyr (SEQ ID NO:4),Ser-Gly-Gly-Phe (SEQ ID NO:5), Ser-Gly-Gly-dPhe (SEQ ID NO:6),Ser-Gly-Gly-Tyr (SEQ ID NO:7), Ser-Gly-Gly-dTyr (SEQ ID NO:8), orO-Benzlyoxy. Contemplated equivalents include aromatic functional groupsat R₁ which are not part of tyrosine or phenylalanine.

The function of R₂ is to form bonds with R₁ and R₃ as well as to formbonds with R₄ which cyclicize or otherwise conformationally restrict themolecule. Bonds between R₂ and R₄ cyclicize the molecule and therebymaintain R₂—R₃—R₄, and, specifically R₃, in a constrained conformationwhich produces the specific biologically active surface that has anaffinity for and interacts with p185. Further, in such an arrangement R₁becomes an exocyclic portion of the peptide. Accordingly, R₂ may be anymoiety capable of forming bonds with R₄ as well as R₁ and R₃. R₂ ispreferably an amino acid residue, most preferably cysteine. When both R₂and R₄ are cysteine, the disulfide bonds form between the two cysteinescyclicize the molecule. It is contemplated that R₂ may any moiety that,together with R₄, will allow for the cyclization of the portion of themolecule that includes R, —R₂—R₃—R₄—R₅ while rendering R₁ and R₅exocyclic portions of the peptide. Those having ordinary skill in theart can readily prepare peptides according to the present invention inwhich R₂ and R₄ are moieties capable of forming bonds to each other. Thecyclization of linear peptides using disulfide bonds betweennon-adjacent cysteines is well known. Similarly, other non-adjacentamino acid residues may be linked to cyclicize a peptide sequence andthe means to do so are similarly well known. Other methods ofcyclization include those described by Di Blasio, et al., (1993)Biopolymers, 33:1037-1049; Wood, et al., (1992) J. Pep. Prot. Res.,39:533-539; Saragovi, et al., (1992) Immunomethods, 1:5-9; Saragovi, etal., (1991) Science, 253:792-795; Manning, et al., (1993) Reg. Peptides,45:279-283; Hruby, (1993) Biopolymers, 33:10731082; Bach, et al., (1994)New Adv. Peptidomimetics Small Mol. Design, 1:1-26; and Matsuyama, etal., (1992) J. Bacteriol., 174:1769-1776, each of which are incorporatedherein by reference.

R₃ is the active region of the compounds according to this aspect of theinvention. In compounds of the invention, the functional groups of theactive region are in a conformation which places them in a particularthree dimensional arrangement that allows them to interact with theamino acids and functional groups thereon of p185 and to bind to p185through such interactions. In peptide mimetics, the functional groupsare provided in the active three dimensional arrangement but areconnected to modified or different backbones.

In some preferred embodiments, R₃ is DGFYA (SEQ ID NO:9), DGYYA (SEQ IDNO: 10), DGWYA (SEQ ID NO: 11), DGHYA (SEQ ID NO: 12), DGdFYA (SEQ IDNO: 13), DGdYYA (SEQ ID NO: 14), DGdWYA (SEQ ID NO: 15), DGdHYA (SEQ IDNO: 16), DGFYdA (SEQ ID NO: 17), DGYYdA (SEQ ID NO: 18), DGWYdA (SEQ IDNO: 19), DGHYdA (SEQ ID NO:20), DGdFYdA (SEQ ID NO:21), DGdYYdA (SEQ IDNO:22), DGdWYdA (SEQ ID NO:23), DgdHYdA (SEQ ID NO:24), DGFYG (SEQ IDNO:25), DGYYG (SEQ ID NO:26), DGWYG, (SEQ ID NO:27) DGHYG (SEQ IDNO:27), DGdFYG (SEQ ID NO:29), DGdYYG (SEQ ID NO:30), DGdWYG (SEQ IDNO:31), DgdHYG (SEQ ID NO:32), DGFYP (SEQ ID NO:33), DGYYP (SEQ IDNO:34), DGWYP (SEQ ID NO:35), DGHYP (SEQ ID NO:36), DGdFYP (SEQ IDNO:37), DGdYYP (SEQ ID NO:38), DGdWYP (SEQ ID NO:39), DGdHYP (SEQ IDNO:40), DGFYdP (SEQ ID NO:41), DGYYdP (SEQ ID NO:42), DGWYdP (SEQ IDNO:43), DGHYdP (SEQ ID NO:44), DGdFYdP (SEQ ID NO:45), DGdYYdP (SEQ IDNO:46), DGdWYdP (SEQ ID NO:47), or DGdHYdP (SEQ ID NO:48).

The function of R₄ is to form bonds with R₂ which cyclicize or otherwiseconformationally restrict the molecule. Bonds between R₄ and R₂cyclicize the molecule and thereby maintain R₂—R₃—R₄, and, specificallyR₃, in a constrained conformation which produces the specificbiologically active surface that has an affinity for and interacts withp185. Accordingly, R₄ may be any moiety capable of forming bonds with R₂as well as R₃ and R₅. R₄ is preferably an amino acid residue, mostpreferably cysteine. When both R₅ and R₂ are cysteine, disulfide bondsformed between the two cysteines cyclicizes the molecule. It iscontemplated that R₄ may be any moiety that, together with R₂, willallow for the cyclization of the molecule. Those having ordinary skillin the art can readily prepare peptides according to the presentinvention in which R₂ and R₄ are moieties capable of forming bonds toeach other. The cyclization of linear peptides using disulfide bondsbetween non-adjacent cysteines is well known. Similarly, othernon-adjacent amino acid residues may be linked to cyclicize a peptidesequence and the means to do so are similarly well known. Other methodsof cyclization include those described by Di Blasio, et al., (1993)Biopolymers, 33:1037-1049; Wood, et al., (1992) J. Pep. Prot. Res.,39:533-539; Saragovi, et al., (1992) Immunomethods, 1:5-9; Saragovi, etal., (1991) Science, 253:792-795; Manning, et al., (1993) Reg. Peptides,45:279-283; Hruby, (1993) Biopolymers, 33:1073-1082; Bach, et al.,(1994) New Adv. Peptidomimetics Small Mol. Design, 1:1-26; andMatsuyama, et al., (1992) J. Bacteriol., 174:1769-1776, each of whichare incorporated herein by reference.

The primary function of R₅ in compounds of the present invention arisesfrom the presence of at least one amino acid that contains an aromaticgroup: i.e. the presence of tyrosine or phenylalanine. The presence ofthe aromatic amino acid at position R₅ results in an increase affinityof the peptide to p185 and an attendant increase in activity of thecompound. In embodiments where additional amino acid residues arepresent, they can present the aromatic amino acid in a more effectiveposition to further increase the affinity and activity of the compound.Additional amino acids that may be present must not eliminate the effectthat the aromatic amino acid has on affinity or activity. Examples ofamino acid sequences which may be used as R₅ are disclosed in U.S. Pat.No. 6,100,377. In some embodiments, the additional amino acids arepresent as a site for linkage to detectable labels or moieties. In someembodiments, the additional amino acids are present as a site fordimerization with other peptides; either for formation of homodimerswith each other or heterodimers with other peptides In some preferredembodiments, R₅ is 1-13 amino acids. In some preferred embodiments, R₅is 12 amino acids. In some preferred embodiments, R₅ is 11 amino acids.In some preferred embodiments, R₅ is 10 amino acids. In some preferredembodiments, R₅ is 9 amino acids. In some preferred embodiments, R₅ is 8amino acids. In some preferred embodiments, R₂ is 7 amino acids. In somepreferred embodiments, R₅ is 6 amino acids. In some preferredembodiments, R₅ is 5 amino acids. In some preferred embodiments, R₂ is 4amino acids. In some preferred embodiments, R₅ is 3 amino acids. In somepreferred embodiments, R₅ is 2 amino acids. In some preferredembodiments, R₅ is 1 amino acid. In some preferred embodiments, R₅ isselected from the group consisting of YMDV (SEQ ID NO:49), dYMDV (SEQ IDNO:50), FMDV (SEQ ID NO:51), dFMDV (SEQ ID NO:52) YKDV (SEQ ID NO:53),dYKDV (SEQ ID NO:54), FKDV (SEQ ID NO:55), dFKDV (SEQ ID NO:56), YMDVK(SEQ ID NO:57), dYMDVK (SEQ ID NO:58), FMDVK, (SEQ ID NO:59) dFMDVK (SEQID NO:60), YKDVK (SEQ ID NO:61), dYKDVK (SEQ ID NO:62), FKDVK (SEQ IDNO:63), dFKDVK (SEQ ID NO:64), YMDVG (SEQ ID NO:65), dYMDVG (SEQ IDNO:66), FMDVG (SEQ ID NO:67), dFMDVG (SEQ ID NO:68), YKDVG (SEQ IDNO:69), dYKDVG (SEQ ID NO:70), FKDVG (SEQ ID NO:71), dFKDVG (SEQ IDNO:72), YMDVKG (SEQ ID NO:73), dYMDVKG (SEQ ID NO:74), FMDVKG (SEQ IDNO:75), dFMDVKG (SEQ ID NO:76), YKDVKG (SEQ ID NO:77), dYKDVKG (SEQ IDNO:78), FKDVKG (SEQ ID NO:79), dFKDVKG (SEQ ID NO:80), YMDVGG (SEQ IDNO:81), dYMDVGG (SEQ ID NO:82), FMDVGG (SEQ ID NO:83), dFMDVGG (SEQ IDNO:84), YKDVGG (SEQ ID NO:85), dYKDVGG (SEQ ID NO:86), FKDVGG (SEQ IDNO:87), dFKDVGG (SEQ ID NO:88), YMDVKGG (SEQ ID NO:89), dYMDVKGG (SEQ IDNO:90), FMDVKGG (SEQ ID NO:91), dFMDVKGG (SEQ ID NO:92), YKDVKGG (SEQ IDNO:93), dYKDVKGG, (SEQ ID NO:94) FKDVKGG (SEQ ID NO:95), dFKDVKGG (SEQID NO:96), YMDVGGS (SEQ ID NO:97), dYMDVGGS (SEQ ID NO:98), FMDVGGS (SEQID NO:99), dFMDVGGS (SEQ ID NO:100), YKDVGGS (SEQ ID NO:101), dYKDVGGS(SEQ ID NO:102), FKDVGGS (SEQ ID NO: 103), dFKDVGGS (SEQ ID NO: 104),YMDVKGGS (SEQ ID NO:105), dYMDVKGGS (SEQ ID NO:106), FMDVKGGS (SEQ IDNO:107), dFMDVKGGS (SEQ ID NO: 108), YKDVKGGS (SEQ ID NO: 109),dYKDVKGGS (SEQ ID NO: 110), FKDVKGGS (SEQ ID NO:111), or dFKDVKGGS (SEQID NO: 112). In some preferred embodiments, R₅ is selected from thegroup consisting of Y⁻R₅₁, dY-R₅₁, F-R₅₁, or dF-R₅₁, wherein R₅₁, is anylong aliphatic chain of d and/or 1 amino acids. In some preferredembodiments, R₅ is selected from the group consisting of Y⁻R₅₁, dY-R₅₁,F⁻R₅₁, or dF⁻R₅₁, wherein R₅₁, is an amino acid chain comprising up to12 amino acids independently selected from the group consisting of Lys,Leu and Ile.

In some preferred embodiments, R₁ and R₅ collectively contain bothtyrosine and phenylalanine. That is, if R₁ comprises tyrosine then R₅comprises phenylalanine and if R₁ comprises phenylalanine then R₅comprises tyrosine. In some preferred embodiments, R₁ and R₅ do not bothcontain tyrosine or phenylalanine. That is, if R₁ comprises tyrosine andnot phenylalanine then R₅₅ comprises phenylalanine and not tyrosine andif R₁ comprises phenylalanine and not tyrosine then R₅ comprisestyrosine and not phenylalanine.

In some preferred embodiments, R₁, R₂, R₃, R₄ and R₅, taken together,are less than 20 amino acids. In some preferred embodiments, R₁, R₂, R₃,R₄ and R₅, taken together, are 19 amino acids or less. In some preferredembodiments, R₁, R₂, R₃, R₄ and R₅, taken together, are less than 18amino acids. In some preferred embodiments, R₁, R₂, R₃, R₄ and R₅, takentogether, are 17 amino acids. In some preferred embodiments, R₁, R₂, R₃,R₄ and R₅, taken together, are less than 16 amino acids. In somepreferred embodiments, R₁R₂, R₃, R₄ and R₅, taken together, are lessthan 15 amino acids. In some preferred embodiments, R₁, R₂, R₃, R₄ andR₅, taken together, are 14 amino acids. In some preferred embodiments,R₁R₂, R₃, R₄ and R₅, taken together, are 13 amino acids. In somepreferred embodiments, R₁R₂, R₃, R₄ and R₅, taken together, are 12 aminoacids. In some preferred embodiments, R₁ R₂, R₃, R₄ and R₅, takentogether, are 11 amino acids. In some preferred embodiments, R₁ R₂, R₃,R₄ and R₅, taken together, are 10 amino acids.

In some embodiments, the peptide is selected from the group consistingof:

YCDGFYACYMDV-NH₂, (SEQ ID NO: 113) YCDGFYACYMDV-OH, (SEQ ID NO: 114)GYCDGFYACYMDV, (SEQ ID NO: 115) GGYCDGFYACYMDV, (SEQ ID NO: 116)GGGYCDGFYACYMDV, (SEQ ID NO: 117) dFCDGFYACdYMDV-NH₂, (SEQ ID NO: 118)dFCDGFYACdYMDV-OH, (SEQ ID NO: 119) GdFCDGFYACdYMDV, (SEQ ID NO: 120)GGdFCDGFYACdYMDV, (SEQ ID NO: 121) GGGdFCDGFYACdYMDV, (SEQ ID NO: 122)FCDGFYACYMDVK-NH₂, (SEQ ID NO: 123) dFCDGFYACdYMDVK-OH, (SEQ ID NO: 124)GdFCDGFYACdYMDVK, (SEQ ID NO: 125) GGdFCDGFYACdYMDVK, (SEQ ID NO: 126)GGGdFCDGFYACdYMDVK, (SEQ ID NO: 127) FCDGFYACYKDV-NH₂, (SEQ ID NO: 128)FCDGFYACYKDV-OH, (SEQ ID NO: 129) GFCDGFYACYMDV, (SEQ ID NO: 130)GGFCDGFYACYKDV, (SEQ ID NO: 131) GGGFCDGFYACYKDV, (SEQ ID NO: 132)GFCDGFYACYMDV-NH₂, (SEQ ID NO: 133) GFCDGFYACYMDV-OH, (SEQ ID NO: 134)GFCDGFYACYMDVG, (SEQ ID NO: 135) GFCDGFYACdYMDVGG, (SEQ ID NO: 136) andGFCDGFYACdYMDVGGG. (SEQ ID NO: 137)

In some embodiments, the peptide is according to Formula I exceptYCDGFYACYMDV-NH₂ (SEQ ID NO:113), dFCDGFYACdYMDV-NH₂ (SEQ ID NO: 118),FCDGFYACYMDVK-NH₂ (SEQ ID NO: 123), FCDGFYACYKDV-OH (SEQ ID NO: 129),and GFCDGFYACYMDV-OH (SEQ ID NO: 134).

According to some embodiments, the present invention provides peptideshaving Formula II:R₆—R₇—R₈—R₉—R₁₀  (II)wherein

-   -   R₆ is 1-4 amino acid residues including at least one of tyrosine        or phenylalanine;    -   R₇ is cysteine;    -   R₈ is 5-7 amino acids;    -   R₉ is cysteine;    -   R₁₀ is 1-13 amino acid residues and at least one of which is        tyrosine or phenylalanine;

wherein: R₆, R₇, R₈, R₉, and R₁₀ taken together, are 20 amino acids orless; and

-   -   R₈ has the formula        R₈₁—R₈₂—R₈₃        wherein        -   R₈₁ is glycine-aspartic acid, proline-aspartic acid, or            aspartic acid;        -   R₈₂ is glycine, proline or proline-proline; and        -   R₈₃ is phenylalanine-tyrosine-alanine;            and the carboxy terminus of R₁₀ is either amidated or            hydroxylated.

In some preferred embodiments, R₆ consists of Phe, dPhe, Tyr, dTyr,Gly-Phe, Gly-dPhe, Gly-Tyr, Gly-dTyr, Ala-Phe, Ala-dPhe, Ala-Tyr,Ala-dTyr, Lys-Phe, Lys-dPhe, Lys-Tyr, Lys-dTyr, Gly-Gly-Phe,Gly-Gly-dPhe, Gly-Gly-Tyr, Gly-Gly-dTyr, Gly-Gly-Gly Phe (SEQ ID NO: 1),Gly-Gly-Gly-dPhe (SEQ ID NO:2), Gly-Gly-Gly-Tyr (SEQ ID NO:3),Gly-Gly-Gly-dTyr (SEQ ID NO:4), Ser-Gly-Gly-Phe (SEQ ID NO:5),Ser-Gly-Gly-dPhe (SEQ ID NO:6), Ser-Gly-Gly-Tyr (SEQ ID NO:7), orSer-Gly-Gly-dTyr (SEQ ID NO:8). Contemplated equivalents includearomatic functional groups at R₁ which are not part of tyrosine orphenylalanine. R₆ is preferably phenylalanine in some preferredembodiments.

In some preferred embodiments, R₈ has the formulaR₈₁—R₈₂—R₈₃wherein

-   -   R₈₁ is glycine-aspartic acid, proline-aspartic acid, or aspartic        acid;    -   R₈₂ is glycine, proline or proline-pro line; and R₈₃ is        phenylalanine-tyrosine-alanine. In some preferred embodiments,        R₈ consists of GDGFYA (SEQ ID NO: 138), GDGFYA (SEQ ID NO: 139),        DPFYA (SEQ ID NO: 140), PDGFYA (SEQ ID NO: 141), or DPPFYA (SEQ        ID NO: 142).

In some preferred embodiments, R₁₀ is selected from the group consistingof YMDV (SEQ ID NO:49), dYMDV (SEQ ID NO:50), FMDV (SEQ ID NO:51), dFMDV(SEQ ID NO:52) YKDV (SEQ ID NO:53), dYKDV (SEQ ID NO:54), FKDV (SEQ IDNO:55), dFKDV (SEQ ID NO:56), YMDVK (SEQ ID NO:57), dYMDVK (SEQ IDNO:58), FMDVK, (SEQ ID NO:59) dFMDVK (SEQ ID NO:60), YKDVK (SEQ IDNO:61), dYKDVK (SEQ ID NO:62), FKDVK (SEQ ID NO:63), dFKDVK (SEQ IDNO:64), YMDVG (SEQ ID NO:65), dYMDVG (SEQ ID NO:66), FMDVG (SEQ IDNO:67), dFMDVG (SEQ ID NO:68), YKDVG (SEQ ID NO:69), dYKDVG (SEQ IDNO:70), FKDVG (SEQ ID NO:71), dFKDVG (SEQ ID NO:72), YMDVKG (SEQ IDNO:73), dYMDVKG (SEQ ID NO:74), FMDVKG (SEQ ID NO:75), dFMDVKG (SEQ IDNO:76), YKDVKG (SEQ ID NO:77), dYKDVKG (SEQ ID NO:78), FKDVKG (SEQ IDNO:79), dFKDVKG (SEQ ID NO: 80), YMDVGG (SEQ ID NO: 81), dYMDVGG (SEQ IDNO: 82), FMDVGG (SEQ ID NO:83), dFMDVGG (SEQ ID NO:84), YKDVGG (SEQ IDNO:85), dYKDVGG (SEQ ID NO:86), FKDVGG (SEQ ID NO:87), dFKDVGG (SEQ IDNO:88), YMDVKGG (SEQ ID NO:89), dYMDVKGG (SEQ ID NO:90), FMDVKGG (SEQ IDNO:91), dFMDVKGG (SEQ ID NO:92), YKDVKGG (SEQ ID NO:93), dYKDVKGG, (SEQID NO:94) FKDVKGG (SEQ ID NO:95), dFKDVKGG (SEQ ID NO:96), YMDVGGS (SEQID NO:97), dYMDVGGS (SEQ ID NO:98), FMDVGGS (SEQ ID NO:99), dFMDVGGS(SEQ ID NO: 100), YKDVGGS (SEQ ID NO: 101), dYKDVGGS (SEQ ID NO: 102),FKDVGGS (SEQ ID NO: 103), dFKDVGGS (SEQ ID NO: 104), YMDVKGGS (SEQ IDNO:105), dYMDVKGGS (SEQ ID NO:106), FMDVKGGS (SEQ ID NO:107), dFMDVKGGS(SEQ ID NO: 108), YKDVKGGS (SEQ ID NO: 109), dYKDVKGGS (SEQ ID NO: 110),FKDVKGGS (SEQ ID NO: 111), or dFKDVKGGS (SEQ ID NO: 112). In somepreferred embodiments, R₁₀ is: YMDV (SEQ ID NO:49).

In some embodiments, the peptide is selected from the group consistingof: FCGDGFYACYMDV-NH₂ (SEQ ID NO: 143), FCGDGFYACYMDV-OH (SEQ ID NO:144), FCDGFYACYMDV-NH₂ (SEQ ID NO: 145), FCDGFYACYMDV-OH (SEQ ID NO:146), FCDPFYACYMDV-NH₂ (SEQ ID NO: 147), FCDPFYACYMDV-OH (SEQ ID NO:147), FCPDGFYACYMDV-NH₂ (SEQ ID NO: 148), FCPDGFYACYMDV-OH (SEQ ID NO:149), FCDPPFYACYMDV-NH₂ (SEQ ID NO: 150), and FCDPPFYACYMDV-OH (SEQ IDNO: 151). In some embodiments, the peptide is according to Formula IIexcept FCGDGFYACYMDV-NH₂ (SEQ ID NO: 143), FCGDGFYACYMDV-OH (SEQ ID NO:144), FCDGFYACYMDV-NH₂ (SEQ ID NO: 145), FCDGFYACYMDV-OH (SEQ ID NO:146), FCDPFYACYMDV-NH₂ (SEQ ID NO:147), FCDPFYACYMDV-OH (SEQ ID NO:147), FCPDGFYACYMDV-NH₂ (SEQ ID NO: 148), FCPDGFYACYMDV-OH (SEQ ID NO:149), FCDPPFYACYMDV-NH₂ (SEQ ID NO: 150), and FCDPPFYACYMDV-OH (SEQ IDNO: 151).

Those having ordinary skill in the art can readily construct moleculesaccording Formula I or Formula II and determine whether or not thecompounds are active as p185 binding compounds which prevent andeliminate the p185-mediated transformation phenotype.

The peptides of the invention may be dimerized, with each other to formhomodimers or with other compounds including compounds of the inventionto form heterodimers. In preferred dimers, the residues involved in thechemical bound which links the monomers is in the R₁ position of thecompounds of the invention.

The compositions used in the method of treating, preventing or imagingtumors or quantifying p185 may comprise mimetics instead of peptides. Asused herein, the term “Mimetics” is used to refer to compounds whichmimic the activity of peptide. Mimetics are non-peptides but maycomprise amino acids linked by non-peptide bonds. Parent applicationU.S. Pat. No. 5,677,637 issued Jun. 10, 1997 and parent applicationsthereof, all of which are incorporated herein by reference, containdetailed guidance on the production of mimetics. Briefly, the threedimensional structure of the peptides which specifically interacts withthe three dimensional structure of the p185 is duplicated by a moleculethat is not a peptide.

The compounds of the invention may be used to treat individualssuffering from p185-associated tumors. According to one aspect of theinvention, compounds are administered to individuals suspected of havingp185 tumors. Those having ordinary skill in the art can readilydetermine whether an individual may have a tumor likely to be ap185-associated tumor. Biopsy protocols can be performed to identifytumor samples and determine whether or not they are p185 associatedtumors. The diagnostic/characterization protocol described above may beused in the characterization and determination of p185 levels present oncell samples.

The compounds of the invention may be used to prevent the occurrence ofp185 associated tumors in individuals susceptible to p185-associatedtumors. According to one aspect of the invention, compounds areadministered prophylactically to individuals susceptible to developingp185 tumors. Those having ordinary skill in the art can readilydetermine whether an individual may be susceptible to p185 associatedtumors. The invention is particularly useful in high risk individualswho, for example, have a family history of erbB-2-associated cancer orshow a genetic predisposition. Additionally, the present invention isparticularly useful to prevent patients who have had erbB-2-associatedtumors removed by surgical resection or who have been diagnosed ashaving erbB-2-associated cancer in remission.

Methods of the present invention comprise administering a single ormultiple doses of the compounds of the invention. Preferred for humanpharmaceutical use are pharmaceutical compositions that comprise thecompounds of the present invention in combination with apharmaceutically acceptable carrier or diluent.

The pharmaceutical compositions of the present invention may beadministered by any means that enables the active agent to reach theagent's site of action in the body of a mammal. In the case of thepeptides of the invention, the primary focus is the ability to reach andbind with cellular p185. Because proteins are subject to being digestedwhen administered orally, parenteral administration, i.e., intravenous,subcutaneous, intramuscular, would ordinarily be used to optimizeabsorption. These small compact forms are resistant to many proteasesand should be orally available.

In one aspect, the compounds of the present invention are administeredin combination with other cancer therapeutics used to treatc-erbB-2-associated tumors, such as Herceptin or tamoxifen.

In addition to pharmaceutical compositions which comprise compounds ofthe invention, alone or in combination with other cancer therapeutics,therapeutic and diagnostic pharmaceutical compositions of the presentinvention include conjugated compounds specifically targeted to p185.The pharmaceutical compositions which comprise conjugated compositionsof the present invention may be used to diagnose or treat individualssuffering from p185-associated cancer.

One aspect of the present invention relies upon the use of a compound ofthe invention conjugated to a detectable and/or cytotoxic agent. Inconjugated compositions, the compound of the invention delivers theactive agent to cells that have p185. Thus, cells which overexpress p185will be contacted with more active agents than other cells. The activeagent is useful to image, inhibit proliferation of and/or kill the cell.According to one aspect of the present invention, the active agent is atherapeutic agent or an imaging agent. In a preferred embodiment, theimaging agent is ^(99m)Tc, chemically conjugated to the peptides of thepresent invention using, e.g., DOTA and DTPA.

Some chemotherapeutic agents may be used as active agents and conjugatedwith compounds of the invention. Chemotherapeutics are typically, smallchemical entities produced by chemical synthesis and include cytotoxicdrugs, cytostatic drugs as well as compounds which affects cells inother ways such as reversal of the transformed state to a differentiatedstate or those which inhibit cell replication. Examples ofchemotherapeutics include, but are not limited to: methotrexate(amethopterin), doxorubicin (adrimycin), daunorubicin,cytosinarabinoside, etoposide, 5-4 fluorouracil, melphalan,chlorambucil, and other nitrogen mustards (e.g., cyclophosphamide),cis-platinum, vindesine (and other vinca alkaloids), mitomycin andbleomycin.

Active agents may be toxins: complex toxic products of various organismsincluding bacteria, plants, etc. Examples of toxins include but are notlimited to: ricin, ricin A chain (ricin toxin), Pseudomionas exotoxin(PE), diphtheria toxin (DT), Clostridium perfringens phospholipaseC(PLC), bovine pancreatic ribonuclease (BPR), pokeweed antiviral protein(PAP), abrin, abrin A chain (abrin toxin), cobra venom factor (CVF),gelonin (GEL), saporin (SAP), modeccin, viscumin and volkensin. Proteintoxins may be produced using recombinant DNA techniques as fusionproteins which include peptides of the invention. Protein toxins mayalso be conjugated to compounds of the invention by non-peptidyl bonds.

Radioisotopes may be conjugated to compounds of the invention to providecompositions that are useful as therapeutic agents or for imagingprocedures. Examples of radioisotopes which useful in radiation therapyinclude: ⁴⁷Sc, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au,²¹¹At, ²¹²Pb and ²¹²Bi. Example of radioisotopes useful in imagingprocedures include: ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br,⁸¹Rb/^(81M)Sr, ^(87M)Sr, ⁹⁹Tc, ¹¹¹In, ^(113M)I, ¹²³I, ¹²⁵I, ¹²⁷Cs,¹²⁹Cs, ¹³¹I, ¹³²I, ¹⁹⁷Hg, ²⁰³Pb and ²⁰⁶Bi. Preferred radioistopes areTc, particularly ^(99m)Tc, Y, particularly ⁹⁰Y, and ¹⁸F.

Imaging agents are useful in diagnostic procedures as well as theprocedures used to identify the location of p 185 associated tumors.Imaging can be performed by many procedures well-known to those havingordinary skill in the art and the appropriate imaging agent useful insuch procedures may be conjugated to compounds of the invention bywell-known means. Imaging can be performed, for example, byradioscintigraphy, nuclear magnetic resonance imaging (MRI) or computedtomography (CT scan). The most commonly employed radiolabels for imagingagents include radioactive iodine and indium. Imaging by CT scan mayemploy a heavy metal such as iron chelates. MRI scanning may employchelates of gadolinium or manganese. Additionally, positron emissiontomography (PET) may be possible using positron emitters of oxygen,nitrogen, iron, carbon, or gallium.

Radiolabels are conjugated to compounds of the invention by a variety ofwell-known techniques readily performed without undue experimentation bythose having ordinary skill in the art. Radiolabels retain theirradioactivity irrespective of conjugation. Conjugation may beaccomplished directly between the compound and the radioisotope orlinking, intermediate molecular groups may be provided between thecompound and the radioisotope. Crosslinkers are particularly useful tofacilitate conjugation by providing attachment sites for each moiety.Crosslinkers may include additional molecular groups which serve asspacers to separate the moieties from each other to prevent either frominterfering with the activity of the other. Often imaging can be imagedusing fluorescein, which are activated by light. (e.g. fluorescein(green), phycoerythrin (orange), P-E-cyanine-5 (red), P-E-texas red(red), cyanine-3 (orange), cyananine-5 (red), AMCA (ultravioletdetection). Examples of crosslinkers include DOTA/DTPA.

One having ordinary skill in the art may conjugate a compound of theinvention to a chemotherapeutic drug using well-known techniques. Forexample, Magerstadt, M. Antibody Conjugates and Malignant Disease.(1991) CRC Press, Boca Raton, USA, pp. 110 152) which is incorporatedherein by reference, teaches the conjugation of various cytostatic drugsto amino acids of antibodies. Such reactions may be applied to conjugatechemotherapeutic drugs to the compounds of the invention. Compounds ofthe invention such as peptides which have a free amino group may beconjugated to active agents at that group. Most of the chemotherapeuticagents currently in use in treating cancer possess functional groupsthat are amenable to chemical crosslinking directly with proteins. Forexample, free amino groups are available on methotrexate, doxorubicin,daunorubicin, cytosinarabinoside, cis-platin, vindesine, mitomycin andbleomycin while free carboxylic acid groups are available onmethotrexate, melphalan, and chlorambucil. These functional groups, thatis free amino and carboxylic acids, are targets for a variety ofhomobifunctional and heterobifunctional chemical crosslinking agentswhich can crosslink these drugs directly to the single free amino groupof a compound of the invention.

Pharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. They can be administered alone, but are generallyadministered with a pharmaceutical carrier selected on the basis of thechosen route of administration and standard pharmaceutical practice.

The dosage administered will, of course, vary depending upon knownfactors such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adaily dosage of active ingredient can be about 0.001 to 1 grams perkilogram of body weight, in some embodiments about 0.1 to 100 milligramsper kilogram of body weight. Ordinarily dosages are in the range of 0.5to 50 milligrams per kilogram of body weight, and preferably 1 to 10milligrams per kilogram per day. In some embodiments, the pharmaceuticalcompositions are given in divided doses 1 to 6 times a day or insustained release form is effective to obtain desired results.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 1 milligram to about 500 milligrams ofactive ingredient per unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about0.5-95 by weight based on the total weight of the composition.

Because conjugated compounds are specifically targeted to cells withp185, conjugated compounds which comprise chemotherapeutics or toxinsare administered in doses less than those which are used when thechemotherapeutics or toxins are administered as unconjugated activeagents, preferably in doses that contain up to 100 times less activeagent. In some embodiments, conjugated compounds which comprisechemotherapeutics or toxins are administered in doses that contain10-100 times less active agent as an active agent than the dosage ofchemotherapeutics or toxins administered as unconjugated active agents.To determine the appropriate dose, the amount of compound is preferablymeasured in moles instead of by weight. In that way, the variable weightof different compounds of the invention does not affect the calculation.Presuming a one to one ratio of p185-binding compound to active agent inconjugated compositions of the invention, less moles of conjugatedcompounds may be administered as compared to the moles of unconjugatedcompounds administered, preferably up to 100 times less moles.

For parenteral administration, the compound can be formulated as asolution, suspension, emulsion or lyophilized powder in association witha pharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Liposomes and nonaqueous vehicles such as fixedoils may also be used. The vehicle or lyophilized powder may containadditives that maintain isotonicity (e.g., sodium chloride, mannitol)and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

For example, a parenteral composition suitable for administration byinjection is prepared by dissolving 1.5% by weight of active ingredientin 0.9% sodium chloride solution.

According to the present invention, the compound may be administered totissue of an individual by topically or by lavage. The compounds may beformulated as a cream, ointment, salve, douche, suppository or solutionfor topical administration or irrigation. Formulations for such routesadministration of pharmaceutical compositions are well known. Generally,additives for isotonicity can include sodium chloride, dextrose,mannitol, sorbitol and lactose. In some cases, isotonic solutions suchas phosphate buffered saline are used. Stabilizers include gelatin andalbumin. In some embodiments, a vasoconstriction agent is added to theformulation. The pharmaceutical preparations according to the presentinvention are preferably provided sterile and pyrogen free.

One of skill in the art of pharmaceutical formulations, e.g., having anadvanced degree in Pharmaceutics or Pharmaceutical Sciences, can preparea variety of appropriate dosage forms and formulations for thecompositions of the invention with no more than routine experimentation.A number of texts in the field, a,g., Remington's PharmaceuticalSciences and The U.S. Pharmacopoeia/National Formulary, latest editions,provide considerable guidance in this respect.

A pharmaceutically acceptable formulation will provide the activeingredient(s) in proper physical form together with such excipients,diluents, stabilizers, preservatives and other ingredients as areappropriate to the nature and composition of the dosage form and theproperties of the drug ingredient(s) in the formulation environment anddrug delivery system.

The compositions may include additional components to render them moreeffective. For example, a composition of the invention may comprisemultiple anti-p185 compounds. The compositions may include otheranti-cancer agents such as, for example, cis-platin, methotrexate,and/or G-MCSF. Such compositions would be particularly useful foradministration to patients diagnosed and treated for erbB-2-associatedcancer.

Administration Regimen

About 5 μg to 5000 mg of peptide may be administered. In some preferredembodiments, 50, μg to 500 mg of peptide may be administered. In otherpreferred embodiments, 500 μg to 50 mg of peptide may be administered.In a preferred embodiment, 5 mg of peptide is administered.

Prophylacetic compositions may be administered by an appropriate routesuch as, for example, by oral, intranasal, intramuscular,intraperitoneal or subcutaneous administration. In some embodiments,intravenous administration is preferred.

Subsequent to initial administration, individuals may be boosted byreadministration. In some preferred embodiments, multipleadministrations are performed.

EXAMPLES

The present invention is further described by means of the example,presented below. The use of such an example is illustrative only and inno way limits the scope and meaning of the invention or of anyexemplified term. Likewise, the invention is not limited to anyparticular preferred embodiments described herein. Indeed, manymodifications and variations of the invention will be apparent to thoseskilled in the art upon reading this specification and can be madewithout departing from its spirit and scope. The invention is thereforeto be limited only by the terms of the appended claims along with thefull scope of equivalents to which the claims are entitled.

Example 1

A peptide is selected from the group consisting of: YCDGFYACYMDV-NH₂(SEQ ID NO: 113), YCDGFYACYMDV-OH (SEQ ID NO: 114), GYCDGFYACYMDV (SEQID NO: 115), GGYCDGFYACYMDV (SEQ ID NO: 116), GGGYCDGFYACYMDV (SEQ IDNO: 117), dFCDGFYACdYMDV-NH₂ (SEQ ID NO: 118), dFCDGFYACdYMDV-OH (SEQ IDNO:119), GdFCDGFYACdYMDV (SEQ ID NO: 120), GGdFCDGFYACdYMDV (SEQ IDNO:121), GGGdFCDGFYACdYMDV (SEQ ID NO: 122), FCDGFYACYMDVK-NH₂ (SEQ IDNO: 123), dFCDGFYACdYMDVK-OH (SEQ ID NO: 124), GdFCDGFYACdYMDVK (SEQ IDNO: 125), GGdFCDGFYACdYMDVK (SEQ ID NO: 126), GGGdFCDGFYACdYMDVK (SEQ IDNO: 127), FCDGFYACYKDV-NH₂ (SEQ ID NO: 128), FCDGFYACYKDV-OH (SEQ ID NO:129), GFCDGFYACYKDV (SEQ ID NO: 130), GGFCDGFYACYKDV (SEQ ID NO: 131),GGGFCDGFYACYKDV (SEQ ID NO: 132), GFCDGFYACYMDV-NH₂ (SEQ ID NO: 133),GFCDGFYACYMDV-OH (SEQ ID NO: 134), GFCDGFYACYMDVG (SEQ ID NO:135),GFCDGFYACdYMDVGG (SEQ ID NO: 136), and GFCDGFYACdYMDVGGG (SEQ ID NO:137) and conjugated to radioisotopes of Tc, particularly ^(99M)Tc, Y,particularly ⁹⁰Y, and ¹⁸F using as linkers between the radionuclides andpeptides, DOTA/DTPA or DOTA/DTPA-glycine, DOTA/DTPA-glycine-glycine, orDOTA/DTPA-glycine-glycine-glycine.

Example 2

A peptide is selected from the group consisting of. FCGDGFYACYMDV-NH₂(SEQ ID NO: 143), FCGDGFYACYMDV-OH (SEQ ID NO: 144), FCDGFYACYMDV-NH₂(SEQ ID NO: 145), FCDGFYACYMDV-OH (SEQ ID NO: 146), FCDPFYACYMDV-NH₂(SEQ ID NO: 147), FCDPFYACYMDV-OH (SEQ ID NO: 147), FCPDGFYACYMDV-NH₂(SEQ ID NO: 148), FCPDGFYACYMDV-OH (SEQ ID NO: 149), FCDPPFYACYMDV-NH₂(SEQ ID NO: 150), and FCDPPFYACYMDV-OH (SEQ ID NO: 151). In someembodiments, the peptide is according to Formula II exceptFCGDGFYACYMDV-NH, (SEQ ID NO: 143), FCGDGFYACYMDV-OH (SEQ ID NO: 1. 44),FCDGFYACYMDV-NH₂(SEQ ID NO: 145), FCDGFYACYMDV-OH (SEQ ID NO: 146),FCDPFYACYMDV-NH, (SEQ ID NO: 147), FCDPFYACYMDV-OH (SEQ ID NO: 147),FCPDGFYACYMDV-NH₂ (SEQ ID NO: 148), FCPDGFYACYMDV-OH (SEQ ID NO: 149),FCDPPFYACYMDV-NH₂, (SEQ ID NO: 150), and FCDPPFYACYMDV-OH (SEQ ID NO:151) and conjugated to radioisotopes of Tc, particularly ^(90M)Tc, Y,particularly ⁹⁰Y, and 18F using as linkers between the radionuclides andpeptides, DOTA/DTPA or DOTA/DTPA-glycine, DOTA/DTPA-glycine-glycine, orDOTA/DTPA-glycine-glycine-glycine.

Example 3

Overexpression of the HER2 receptor is observed in about 30% of breastand ovarian cancers and is often associated with an unfavorableprognosis. An anti-HER2 peptide (AHNP) based on the structure of theCDR-H3 loop of the anti-HER2 rhumAb 4D5 was designed and showed thatthis peptide can mimic some functions of rhumAb45. The peptide disabledHER2 tyrosine kinases in vitro and in vivo similar to the monoclonalantibody (Park, B.-W. et al. Nat. Biotechnol. 2000, 18, 194-198). AHNPhas been shown to selectively bind to the extracellular domain of theHER2 receptor with a submicromolar affinity in Biacore assays. Inaddition to being a structural and functional mimic of rhumAb 4D5, AHNPcan also effectively compete with the antibody for binding to the HER2receptor indicating a similar binding site for the peptide and theparental antibody. To further develop AHNP as an antitumor agent usefulfor preclinical trials and as a radiopharmaceutical to be used for tumorimaging, a number of derivatives of AHNP have been designed.Structure-fraction relationships have been studied using surface plasmonresonance technology. Some of the AHNP analogues have improved bindingproperties, solubility, and cytotoxic activity relative to AHNP.Residues in the exocyclic region of AHNP appear to be essential forhigh-affinity binding. Kinetic and equilibrium analysis ofpeptide-receptor binding for various AHNP analogues revealed a strongcorrelation between peptide binding characteristics and their biologicalactivity. For AHNP analogues, dissociation rate constants have beenshown to be better indicators of peptide biological activity thanreceptor-binding affinities. The well-documented antibody effects can bemimiced in tumor therapy by much smaller antibody-based cyclic peptideswith potentially significant therapeutic advantages. Strategies used toimprove binding properties of rationally designed AHNP analogues arediscussed.

Introduction

HER2 (neu, c-erbB2) is a member of the epidermal growth factor (EGFR) orHER family of tyrosine kinase receptors that also includes HER 1 (EGFR,c-erbB 1, HER3 (c-erbB3), and HER4 (c-erbB4). Amplification of HER2 geneand overexpression of HER2 protein has been found in breast and ovariancancers, as well as tumors of the lung, salivary gland, kidney, andbladder. Greater expression of HER2 on transformed cells than on normalepithelial tissues allows selective targeting of tumor cells usingvarious strategies. See references 1-14 listed below)

Recently, some progress has been made in the development of monoclonalantibody-based therapeutics targeting tumor cell surface antigens (seereference 15 listed below for a recent review). The anti-HER2 antibody“trastuzumab” (Herceptin; Genentech, San Francisco) produces objectiveresponses in some patients with advanced breast cancer showingoverexpressed HER2 oncoprotein. The antibody has been shown toantagonize the constitutive growth-signaling properties of the HER2system, enlist immune cells to attack and kill the tumor target, andaugment chemotherapy-induced cytotoxicity. (See reference 16 below.)Another important application of antibodies that has been extensivelydeveloped over the past two decades is tumor imaging by numerousanticancer antibodies against various molecular targets including breastcancer imaging. (See reference 17 below.)

Application of intact antibody molecules as therapeutic or diagnosticmolecules remains limited, since they may cause an immune response andhave little tumor penetration and high background noise. One of the waysto overcome the limitation of therapeutic macromolecules is to develop asmall molecule. A promising approach is to design small peptides derivedfrom the antigen-binding site of antibodies. Historically, fewtherapeutic peptide products have been used in the clinic because of thedifficulties with delivery, stability, and above all, withcost-effective and reliable peptide manufacture. However, recentprogress in high quantity peptide synthesis, as well as in screening andoptimization of peptide leads, has resulted in an explosion in thenumber of candidate peptides and a renewed interest in their commercialdevelopment. (See reference 18 below.)

Since complementarity-determining regions (CDRs) of antibodies mediatetheir high affinity binding and specificity to antigens (See reference19 below), peptide analogues of CDRs can be developed for antibodieswith known sequences and structures. (See reference 20-24 below.) Thestrategy of designing CDR-based mimetics has been widely used inrational drug design. (See references 25-39 below.) Although many of thereported peptides display highly specific antigen binding similar to theparenteral antibody, their antigen-binding affinity is in most casessubstantially lower.

Recently, the design of an anti-HER2 peptide mimetic (AHNP, peptide (1),Table 1) derived from the structure of the CDR-H3 loop of the anti-HER2rhumAb 4D5, and demonstrated its in vitro and in vivo activities indisabling HER2 tyrosine kinases similar to the monoclonal antibody wasreported. (See reference 40 below.).

Binding of AHNP has been studies by means of surface plasmon resonance(Biacore) technology. In Biacore experiments, one of the interactingmolecules (termed the ligand) is immobilized on the sensor surface, andthe other interactant (termed the analyte) is continuously flown overthat surface in a micro-flow cell. The interaction between the ligandand the analyte are monitored using a light source that is reflected atthe immobilized chip. Binding of the analyte to the immobilized ligandchanges the resonance angle of the reflected light due to changes in therefractive index of the surface. The response is plotted in real time inthe form of sensorgram curves. The advantage with this approach is itssensitivity, ease of use, and ability to perform experiments with fewmicrogram quantities of proteins and peptides. In addition, the kineticbinding studies reveal association and disassociation rates of theanalyte which may be more relevant for understanding thepharmacokinetics of drug-receptor interactions.

A typical sensorgram for AHNP binding to the HER2 receptor is shown inFIG. 1. Kinetic constants were estimated by global fitting analysis ofthe titration curves to the 1:1 Langmurian interaction model, which gavea ^(k)on of 1.41×10³ M⁻¹s⁻¹, and a ^(k)off of 4.53×10⁻⁴ s⁻. The^(k)off/^(k)on ratio gave a value of 0.32 μM for the dissociationconstant (^(K)D). The curves shown in FIG. 1 were calculated from theexperimentally observed curves by successive subtractions of signalsobtained for the reference surface and averaged signals for the runningbuffer injected under the same conditions as the tested peptide. (Seereferences 41-44 below.) Good fitting of experimental data to thecalculated curves has been observed, suggesting a simplepseudo-first-order interaction between the peptide and the receptor.

A rational design and structure-function analysis of AHNP analogues withimproved pharmacological features that could be used as antitumor agentsand developed into radiopharmaceuticals is discussed here.

Results

A number of anti-HER2/neu peptide mimetic (AHNP) analogues have beenengineered into better therapeutic agents in terms of their bindingproperties, specificity, and solubility. Ways to incorporate a reactiveamino group to conjugate fluorescent and positron emission tomography(PET) agents (see reference 45 below) and studies its effect on bindingto the receptor were explored.

Competition Studies

It was shown earlier that the AHNP peptide can mimic some functions ofthe anti-HER2/neu antibody, rhumAb 4135 in vitro and in vivo (seereference 40 below). The structural mimicry of rhumAb 4D5 by AHNP hasbeen analyzed in terms of binding to the HER2 receptor by means ofcompetition binding between studies between AHNP and the parenteralantibody. To that end, injections of AHNP at variable concentrations(from 0 to 90 μM) were followed by injections of rhumAb 4135 at aconstant 1 nM concentration, using the “Coinject” mode of the Biacoreinstrument. Increase in the amount of preinjected AHNP resulted in asteady inhibition of the antibody binding (FIG. 2) with an apparent IC₅₀of 3.4 EM, indicating overlapping binding sites for AHNP and rhumAb 4135on the surface of HER2.

Design of AHNP Analogues

A number of modifications have been introduced to the sequence of theAHNP peptide to improve its receptor binding and solubility, propertiesthat are important for both major applications: as a therapeutic and asa tumor imaging agent. For the PET studies, ¹⁸F and ⁹⁰Y will be attachedvia an α- or ε-amine group of AHNP. (See reference 45 below.)

Thus, it is necessary to improve AHNP by either increasing theaccessibility of the N-terminal residue or by introducing a Lys residue,which could be readily labeled without diminishing the binding affinity.Two types of changes have been made: (1) addition of polar groups, and(2) mutation of Met to Lys and introduction of D-isomers at the termini.Most of the changes in AHNP were restricted to the N- and C-terminalresidues outside the loop constrained by the disulfide bond to preservethe binding nature of AHNP.

Chronologically, peptide 8 was the first AHNP analogue that was designedbased on the structures of monoclonal antibodies 4D5 and its rathomologue 7.16. To increase rigidity of the cyclic peptide, peptide 2was designed in which one of the R-turn forming Gly residue has beendeleted. (See reference 40 below.) Three other analogues have beendesigned to analyze effects of different substitutions on biologicalactivity, binding properties, and solubility. An AHNP peptide has beendesigned based on the structure of 2 by replacing the C-terminalcarboxylate by an amide group. In 3, the N-terminal Phe of the AHNPpeptide has been replaced with Tyr, and in 4, aromatic residuespositioned before and after the disulfide bond were replaced with theD-amino acid optical isomers. A Lys residue has been included in thesequence of peptide 2 by either replacing Met (resulting in the 6analog) or by placing it as a C-terminal residue (5).

The designed AHNP analogue peptides have been synthesized, cyclized,(except for peptide 9 used as a control), and tested for binding toHER2, biological activity in an MTT assay, and solubility (Table 1).Binding constants including the association (^(k)on) and dissociation(^(k)off) rate constants, and the equilibrium dissociation constant(K_(D)) shown in Table 1 were determined by analyzing dose dependencecurves obtained for each AHNP analogue in a similar fashion as describedfor AHNP. Effects of different substitutions/additions in AHNP sequenceon receptor binding, biological activity, and solubility are summarizedin Table 1.

Kinetic Binding Analysis of the AHNP Analogues

Biacore analysis of interactions between AHNP analogues and immobilizedHER2 has been performed to test effects of the introduced sequencemodifications on the binding affinity and kinetic constants for eachpeptide. Cyclization is known to be an efficient way of constrainingpeptides in a binding-competent conformation. Consistent with that, theaffinity of cyclic peptide 2 for HER2 was more than 5-fold higher thanthat of the linear analogue 9 (Table 1). Restriction of the loop bydeletion of one of the O-turn glycines (transition from 8 to 2) alsoresulted in improvements in both affinity and dissociation kinetics. Theeffect of the charged C-terminal carboxylate group in 2 on bindingproperties has been tested by replacing it with an amide group in AHNP.Elimination of the charged group in the C-terminal tail resulted in morethan 2-fold increase in binding affinity. Similar, but even moredramatic loss of affinity occurred when Lys was introduced as aC-terminal residue in 5 (2.6-fold decrease) and especially when Lys wassubstituted for Met in 6 (8,8-fold decrease). In the latter case, inaddition to the detrimental effect of the C-terminal hydroxyl group, acharge Lys residue replaces a hydrophobic Met residue, which may beimportant for binding. In contrast, significant improvement in binding(more than 2-fold) has been achieved by introducing a polar hydroxylgroup in the N-terminal residue by replacing Phe with Tyr in 3 (PeptideNo. 3 in Table 1: SEQ ID NO: 113). This peptide also had the lowestdissociation rate constant (k_(off)) among all tested AHNP analogues(2.94×10⁻⁴ s⁻¹), which is comparable with the k_(off) of 1.23×10⁻⁴ s⁻¹observed for rhumAb 4D5. (See reference 40 below.)

Solubility.

Four peptides listed in Table 1, 9, 8, 6, and 4 have been shown to havea much higher solubility than the rest of the tested peptides (Table 1).These peptides could be readily dissolved in the PBS buffer, pH 7.4, at1 mg/mL concentration without adjustment of pH. Good solubility has alsobeen observed for the linear form of all tested peptides. In contrast,all other tested peptides had a limited solubility at 1 mg/mL.

Biological Activity of the AHNP Analogues.

Biological activity of AHNP analogues has been evaluated by theirability to inhibit cell proliferation using standard3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tet-razolium bromide) (MTT)assays. (See reference 46 below.) HER2-expressing transformed tumorcells (T6-17) were used for this purpose. (See reference 40 below.) InMTT assays, AHNP inhibited the growth of T6-17 cells, overexpressingtransformed cell line, dose-dependently at concentrations ranging from0.01 to 10 μg/mL. Biological activity of AHNP analogues is shown inTable 1. Each value represents an average of at least four samples.Standard error did not exceed 5% for any of the studied analogues. Awide range of activities has been observed for different peptidesdepending on the nature of introduced modifications. Analogue 3, whichhas an enhanced receptor-binding affinity (K_(D)=150 nM), was also themost active peptide in the MTT assay, showing almost twice the activityof AHNP (Table 1).

Accessibility of the N-terminal Amino Group for Labeling.

Since AHNP analogues can selectively bind to the oncogenic HER2receptor, which is overexpressed in many different forms of cancer,fluorescently or radiolabeled AHNP derivatives could be potentially usedas tumor imaging agents. Therefore, one of the goals was to obtain AHNPanalogues that could be easily modified. Accessibility of differentN-terminal or Lys amino groups for labeling with FITC was estimated bythe HPLC analysis of the peptide-FITC reaction mixture as described inExperimental Procedures. The N-terminal amino group of AHNP has a verylimited accessibility for fluorescent labeling by FITC (about 2-5% ofthe theoretically expected yield). To develop AHNP into aradiopharmaceutical, an active AHNP analogue containing a reactive aminogroup was designed. FITC labeling studies revealed that the N-terminalamino group of AHNP may be inaccessible for labeling. On the basis ofthe molecular model, it appears that the N-terminal aromatic residueburied in the hydrophobic core (FIG. 3A) may hinder access of the bulkymolecule, FITC. To overcome this problem, a more flexible Gly residuewas placed at the N-terminus just before Phel (7). However, this alsodid not significantly improve the degree of labeling (7-10% yield),suggesting that stearic hindrance may still be a factor. Furthermolecular modeling studies showed that 7 can adopt two main low-energyconformations with different orientations of the N-terminal Gly (FIGS.4A and 4B). In one of the conformational states, the N-terminus wasoriented outside the ring created by the disulfide bond and wastherefore solvent-exposed (FIG. 4A). However, in the secondconfirmation, Gly was oriented toward the inside of the ring and wasburied between the ring residues (FIG. 4B). Obviously, the secondconformational state may be predominant in solution for 7, which mayexplain its poor accessibility. Also, since Gly in the secondconformational state is positioned very close to the disulfide bond, itis likely to interfere with peptide cyclization. This may account for anunusually slow cyclization rate that has been observed for 7 relative toother analogues. Cyclization half-time for 7 (about 6 days) is about3-fold longer than a typical half-time, observed for 2 and otheranalogues. Insertion of Gly inside the ring weakened the hydrophobiccore leading to an increased solubility of 7 (Table 1). As expected,both Lys-containing peptides, 6 and 5, were completely accessible forlabeling showing almost qualitative reactivity of their E-amino groupswith FITC. Preliminary binding data obtained with FITC-modified 5indicate that it has a receptor-binding affinity similar to theunlabeled peptide 5.

In Vivo Clearance and Imaging.

AHNP (Peptide 3 in Table 1: SEQ ID NO: 113) labeled with ^(99m)Tc wasused to estimate the blood clearance in nude mice. The mean half-life ofAHNP in the blood is about 50 minutes, and the peptide is completelycleared by about 5 hrs. (FIG. 7). This suggests that AHNP is useful forimaging purposes.

Mice bearing T6-17 xenografted tumors received purified ^(99m)Tc-labeledAHNP. In a preliminary analysis, the ^(99m)Tc, labeled AHNPpreferentially accumulated to the tumor compared to the normal tissue,and the ratio of the % i.d./g is over 3-fold greater on the tumor (seeFIG. 8 and Table 2) than on normal tissue, suggesting that thesemolecules can be engineered for tumor detection.

Enhanced Tumor Cell Growth Inhibition with ANHP and Tamoxifen.

AHNP has previously been demonstrated to enhance growth inhibition ofT6-17 tumor cells in vitro and in vivo in combination withchemotherapeutic agent doxirubicin (Park et al., Nature Biotechnology;18: 194-198 (2000)). Similar to those results, it was demonstrated thatANHP(YCDGFYACYMDV-SEQ ID NO: 113) in combination with tamoxifen enhancedgrowth inhibition of parental MCF-7 p185-expressing and MCF-7/H2p185-overexpressing cells (FIG. 9).

Discussion

Surface plasmon resonance analysis was the method of choice forcharacterization of peptide-receptor binding, since there was interestin not only equilibrium data, but also kinetic parameters of theinteractions. Because of the large number of tested peptides, the onlypractical way for screening was immobilization of HER2 on the chip andinjection of peptides as soluble analytes. Although direct detection ofanalytes smaller than 5000 Da was once considered unfeasible withstandard Biacore protocols (see references 47 and 48 below), recentadvances in the technology, such as higher sensitivity and improvedmicrobluidics, have enabled development of direct binding assays betweenimmobilized proteins and low-molecular-weight analytes includingpeptides (see references 44 and 49-51 below) and organic compounds (seereferences 52-58 below). Reproducible data with a high signal-to-noiseratio have been reported even though the change in molecular mass uponanalyte binding was in some cases as low as 1%. (see references 55 and58 below). In some instances, modifying experimental conditions by usingvery dense ligand surfaces and/or high peptide concentrations with highflow rates was critical for obtaining good signal-to-noise rations (seereferences 55 and 58 below). Accuracy of experiments with low signallevels can be improved by increasing the number of collected datapoints, increasing analyte concentration, and signal averaging of dataderived from repeat sensorgrams. (see reference 44 below). In Biacorestudies of low-molecular-weight-cyclic peptides, highly reproduciblesignals could be obtained after double corrections of data for thereference surface and the running buffer signals.

A large number of antibody-derived peptides have been reported, yetremarkably few of them have been demonstrated to mimic the parental mAbin terms of structure and function. The data show that AHNP not onlyinduce antitumor effects such as its parent antibody, but also sharebinding epitope on the HER2 receptor. This information is veryimportant, since peptides are usually designed to mimic antigen-bindingproperties and therapeutic effects of corresponding mAbs. Althoughpeptide mimetics that bind to receptors are often presumed to be directstructural analogues of the loops that they mimic, and are thereforeexpected to have the same binding sites as the loops, this is not alwaysan obvious fact and has to be proven experimentally. It has been shownthat some peptide mimetics designed to mimic enzyme substrates and evensome natural enzyme inhibitors do not bind in a substrate-like manner.(See references 59-63 below.) For a large number of receptors, analysisof the endogenous peptide and antagonists' binding sites bysite-directed mutagenesis indicated that antagonists and the parentpeptide bind to different subsites. (See references 64-66 below.)Backward binding is a common occurrence which has been exploited todevelop novel inhibitors. (See references 59, 60 and 67 below.)

Although it is shown that AHNP and rhumAb 4D5 interact with the samebinding site on the HER2 surface, it appears that the nature of receptorinteraction with the peptide and the antibody are quite different.Analysis of surface regeneration conditions that are efficient (ornecessary) for the destruction of a ligand-analyte complex can helpprovide insight into the major forces involved in the complex formation.In the rhumAb 4D5-HER2 complex, electrostatic interactions seem to playa predominant role, since the antibody could be easily washed off thereceptor surface by high salt concentrations (4.5 M MgCl₂), but wasresistant to treatment with either detergent (0.2% SDS) or a mixture oforganic solvents. In contrast, the AHNP-HER2 complex was resistant tosalt, but readily dissociated upon addition of either detergent ororganic solvents, suggesting the involvement of hydrophobic interactionsin complex stability. Obviously, aromatic residues at both sides of thedisulfide bond, as well as the hydrophobic residues in the tail of AHNPand its analogues contribute significantly to the overall energy ofbinding, since substitution or modification of these residues adverselyaffects the binding affinity (Table1). Moreover, it is clear that forthe AHNP peptide, in addition to the binding forces inherited from theCDR3 loop of rhumAb 4D5, which obviously direct the peptide to bind tothe CDR3 epitope on the receptor surface, some new complex-stabilizinghydrophobic bonds are formed between the peptide and receptor, which areabsent in the parent antibody.

Poor solubility of peptidomimetics often limits their usefulness astherapeutic agents. The solubility of some AHNP analogues was improvedwithout further loss of the binding characteristics. Molecular modelingwas used to understand the effect: of mutations on solubility. Molecularmodeling AHNP showed formation of a hydrophobic core by Phe 1, Cys2,Phe5, Ala7, Cys8, Tyr9, and Met10 residues upon peptide cyclization(FIG. 3A). Increasing backbone flexibility by adding one more Glyresidue (8) enhanced spatial separation and mobility of the hydrophobicresidues which may have resulted in the improved solubility. Replacementof Met 10 within the improves solubility by reducing the size of thehydrophobic core (FIG. 3B). Phe 1 and Tyr9 at the termini of AHNP formthe center of the core. Replacement of these residues by their D-isomersincreases the separation between them by changing the orientation of thearomatic side chains relative to each other (FIG. 5), which leads to theincreased solubility. Interestingly, when polar groups were introducedin residues outside the hydrophobic core (in 2 and 5), no improvement insolubility has been observed (Table 1), confirming a critical roleplayed by the hydrophobic core in peptide solubility. Another analoguethat displayed a good solubility is 7.

Balancing hydrophilicity and hydrophobicity is critical for highaffinity binding, especially for small molecules. In this study, theeffects of different sequence modifications was tested in the AHNPanalogues on their biological activity and binding; properties.Interestingly, the C-terminal tail region of the studied peptides wasfount to play an important role in receptor binding. In antigen-antibodycomplexes, the CDR loops and framework regions immediately after the CDRloops have been reported to be critical for antigen binding. (Seereferences 36, 37, 68 and 69) However, it is not clear how residues fromthe framework proximal to CDR can play a critical role in binding.

The C-Terminal Met, Asp, and Val residues in AHNP analogues; are derivedfrom the framework region of anti-HER2 antibodies. Replacement of theMet residue in the tail with Lys (6) resulted in a dramatic decrease inbinding affinity by about 1 order of magnitude (Table 1). The effect ofaddition of a Lys residue following the tail sequence 5 on receptorbinding affinity was less dramatic but also significant (2,5-folddecrease, Table 1). Molecular modeling of the AHNP peptide revealed thatthe Met residue and aromatic residues form a hydrophobic core (FIG. 3A).This formation of the hydrophobic core may be critical for receptorbinding, having either enthalpic (formation of hydrophobic bonds withreceptor residues) or entropic (constraining the peptide in an activeconformation) effects, or both. The Met residue is a part of the coreand may also be important for its integrity, which is consistent withthe observed improvement in aqueous solubility of 6 relative to 2 (Table1). These observations suggest that additional interactions of theC-terminal tail residues of the AHNP analogues with the receptor may bepartly compensating for the diminished interface area in thepeptide-receptor complex relative to the antibody-receptor complex.

Structure-activity relationship has been studied for the whole series ofpeptides. Cell growth inhibition activities obtained in the MTT assaysfor each analogue were plotted versus their affinity for HER2, estimatedin the surface plasmon resonance study (;FIG. 6A). A strong correlation(r²=0.89) has been determined between the peptides' receptor affinitiesand their inhibitory effects, suggesting that the observed biologicalactivities are mediated by binding to HER2. As expected, the most activepeptide (3) had the highest binding affinity, while the affinity of theleast active (6) was the lowest among all analogues. Although theoverall correlation was rather strong, some notable deviations from thestraight line have been observed. Thus 2 and 7 peptides have arelatively small (12%) difference in affinities, but much morepronounced difference (41%) in inhibition. Similar discrepancies havebeen detected by comparing AHNP with 4 (9% difference in affinity versus35% difference in inhibitory effect). The biggest inconsistency has beenobserved for the 7 and 5 pair. Although 5 is 27% more active, it bindsto the receptor with a 14% lower affinity than 7 (Table 1).

To test whether these discrepancies could be explained by differences inthe kinetic rate constants, activity data were plotted against thedissociation rate constants (k_(off)) observed in Biacore assays (FIG.6B). Remarkably, inhibitory activity showed an even stronger correlationwith the k_(off) (r²+0.94), than with the K_(D). Comparison of the twoplots (FIGS. 6A and 6B) suggests that stronger inhibitory activityobserved for AHNP, 2, and 5 can be better explained by their slowdissociation rates rather than by their high receptor-bindingaffinities.

Analysis of the drug-receptor dissociation rate is essential for aproper design and interpretation of receptor-binding studies, as well asfor the selection of drug candidates. (See reference 70 and 71 below).For slowly dissociating drugs binding equilibrium cannot be reached inshort-incubation time experiments, thus prevent competitive inhibition.A slow dissociation rate has been shown to play an important role forthe biological activity of the drug. (See references 55 and 70.) Sincerapidly dissociating drugs can reach a competitive binding equilibriumwith the endogenous receptor ligands, they are easily displaced from thereceptor sites by increased concentrations of the ligands, which in mostcases have higher receptor-dinging affinities than the drugs. However,slowly dissociating drugs form inactive receptor-drug complexes whichhave very long half-lives. Even if the overall binding affinity is lowbecause of a slow association rate, these drugs can provoke a permanentreceptor blockade, which cannot be displaced by the endogenous ligands,thus acting as almost nonreversible antagonists. Dissociation rate mightplay an important role in long-term effects of drugs. (See reference 70below). Therefore, analysis based in K_(D) values alone, could overlookpotentially strong inhibitors that have slow binding and slowdissociation rates. (See reference 72 below). Data demonstrates rhumAb4D5 has about 2 orders of magnitude higher receptor-binding affinitythan the AHNP peptides (Table 1). However, this difference is mostly dueto a faster on-rate (higher k_(on)) of the antibody. In terms of thedissociation rate constant k_(off), receptor-binding properties of theoptimized peptides are very similar to those of the antibody. Although abig excess of AHNP is required to inhibit rhumAb 4D5-HER2 interaction(because of the difference in the binding affinity), the optimizedpeptides and the antibody have comparable biological activities in theMTT assay, in line with the observation that k_(off), rather K_(D),determines the biological activity of the AHNP peptides.

Data shows the importance of the dissociation rate constant forbiological activity of AHNP analogues. For this series of peptides,k_(off) has been shown to have a higher predictive value for theexpected inhibitory effects than the dissociation constant (K_(D))traditionally used for these purposes. Since AHNP analogues producetheir biological effects by binding to HER2 and possibly inducing aconformational charge that deactivates the receptor, the data indicatethat increasing the half-life of the inactive peptide-receptor complexesis more efficient for the inhibition of normal receptor functioning thanincreasing the equilibrium concentration of these complexes.

In the HER2 receptor system, AHNP peptides compete with somefast-occurring processes (either binding or conformationalrearrangements) that lead to receptor signaling. Because of their rapidrate, these processes might reoccur each time immediately after thepeptide dissociates from the receptor surface and before it can rebind.By remaining on the receptor surface for prolonged periods of time, AHNPanalogues with low dissociation rates effectively block receptoractivity. The data suggest that high binding affinity does notnecessarily have to be the main goal that determines the success ofstructure-based drug design. As shown for the AHNP mimetics, thedissociation rate constant can be a very important constituent ofpeptides; biological activity. Depending on a drug's mechanism ofaction, a slow k_(off) can compensate for low affinity in certainsituations.

Results from blood clearance analysis of ^(99m)Tc-labeled AHNP suggesteda use for this mimetic for in vivo imaging assays. Subsequent assays innude mice xenografted with p185-expressing tumors (T6-17fibroblast-derived line) showed that AERP preferentially accumulated tothe tumor compared to normal tissue (FIG. 8A-8B). Some accumulation inliver and kidney was also observed. Nevertheless, these studies suggestthat AHNP can be engineered as a useful imaging agent. Furtherrefinements of AHNP are in progress.

Lastly, similar to what was observed for AHNP administered incombination with doxirubicin, a combination of an AHNP analogue (SEQ IDNO: 143) and tamoxifen enhanced in vitro inhibition of proliferation ofp185-expressing and -overexpressing MCF/7 breast carcinoma cells.

In summary, for a number of AHNP analogues, significant improvements inreceptor-binding affinity, solubility, or accessibility for labeling wasachieved by introducing additional hydrophobic or polar groups. Moreimportantly, the optimized analogues showed almost antibody-likedissociation rate constant, which, as shown in structure-activitystudies, is a critical activity-determining parameter for this class ofpeptides. Optimization of both entropic and enthalpic components ofpeptide-receptor binding, performed in this study, has significantlyimproved solubility and binding properties of antibody-derived peptides(including affinities and dissociation rate constants) while retainingthe high specificity typical for a full-size antibody. These analogueswere demonstrated to be useful for in vitro growth inhibition ofp185-expressing tumor cell lines, and in vivo imaging ofxenotransplanted p185-expressing tumor tissue.

Experimental Section

Peptide Synthesis and Cyclization

Linear peptides (95% purity) were ordered from the Protein ChemistryLaboratory, University of Pennsylvania. Peptides purity and identity wasconfirmed by reverse phase high performance liquid chromatography (RPHPLC) and MALDI mass spectrometry, using a time-of-flight massspectrometer (Micromass TofSpec; Micromass Inc., Beverly, Mass.). Thepeptides were cyclized by air oxidation in distilled water adjusted topH 8.0 with (NH₄)₂CO₃ at 0.1 mg/mL and 4° C. Progress of the oxidationwas controlled by measuring amounts of free thiols with5.5′-dithiobis(2-nitrobenzoic) acid (DTNB). Briefly, 0.4 mL of an AHNPpeptide (0.1 mg/mL) and 5 μL of DTNB (20 mM) were added to 0.2 mL of 0.1M sodium phosphate buffer, pH 8.0. Absorbance at 412 nm was measured. ancompared with the linear unoxidized peptides. The cyclized peptides werelyophilized and their purity was analyzed by RP HPLC using a C18semipreparative column (Waters, Milford, Mass.). Typically, purity ofhigher than 95% was obtained for the cyclized peptides. Aliquotes of 1mM stock solutions have been prepared for each peptide and kept at −20°C. to be thawed prior to the binding or bioassay studies. Peptideconcentrations were confirmed by UV spectrophotometry using extinctioncoefficients at 280 nm calculated for each peptide as described inreference 73.

Interaction Studies.

Binding experiments were performed with the surface plasmon resonancebased biosensor instrument Biacore 3000 (Biacore AB, Uppsala, Sweden) at25° C. Recombinant purified HER2 receptor composed of the ectodomain ofHER2 fused to the Fc of human IgG was provided by Dr. Che Law, XcyteTherapeutics, Seattle, Wash. Immobilization of HER2 in the sensorsurface was performed following the standard amine coupling procedureaccording to manufacturer's instructions. Briefly, 35 μL of a solutioncontaining 0.2M N-ethyl-N-(dimethylaminopropyl) carbodiimide (EDC) and0.05 M N-hydroxysuccinimide (NHS) were injected at a flow rate of 5μL/min to activate carboxyl groups on the sensor chip surface. HER2 (40ng/mL in 10 mM NaOAc buffer, pH 5.0) was flowed over the chip surface ata flow rate of 20 μL/min until the desired level bound protein wasreached. Unreacted protein washed out and unreacted activated groupswere blocked by the injection of 35 μL of 1 M ethanolamine at 5 μL/min.The final immobilization response of HER2 was 3500 RU. A referencesurface was generated simultaneously under the same conditions butwithout HER2 injection and used as a blank to correct for instrument andbuffer artifacts. Peptides were injected at variable concentrations at20 μL/min flow rate and binding to the HER2 receptor immobilized on thechip was monitored in real time. Each sensorgram consists of anassociation phase (first 240s), reflecting binding of the injectedpeptide to the receptor, followed by a dissociation phase (300s), duringwhich the running buffer is passed over the chip and the bound peptideis being washed off the receptor surface. In competition studies,peptides were preinjected for 5 min at 20 mL/min at concentrationsranging from 0 to 90 μM. rhumAb 4D5 (Genentech) was then injected for 5min at 1 nM concentration in the “Co-inject” mode. A control cyclicpeptide CD4-M was used in some studies and was shown to be no differentthan blank control.

FITC-Labeling of Peptides.

Two milligrams of each peptide were dissolved in 1 mL of 0.02 M Na₂CO₃NaHCO₃ buffer, pH 9.1, containing 0.02 M NaCl. A total of 0.5 mL of 1%(w/v) fluorescein 5-isothiocyanate (FITC) dissolved in methanol wasadded to the peptide solution, and the reaction mixture was incubated inthe dark for 2 h at room temperature. The reaction was terminated byrapid passage of the reaction mixture through a Sephadex G-10 columnequilibrated with isotonic phosphate-buffered saline, pH 7.4, andfurther purified by C18 reverse-phase HPLC. The purified FITC-labeledpeptides were dried under vacuum. Peptide identity was confirmed byMALDI mass spectroscopy.

MTT Assay.

The MTT assay has been used for measuring cell growth as previouslydescribed in ref 446. Briefly, T6-17 cells were seeded in 96-well platesovernight in DMEM containing 10% FBS (1000 per well). T6-17 is derivedfrom NIH3T3 by overexpressing the human HER2 receptor. Cells werecultured in 100 μL of fresh medium containing 1 μg/mL of peptides for 48h. This incubation time was optimal for measuring inhibitory effects ofdifferent analogues. No improvements in the inhibitory activity could beachieved by increasing the incubation period. A total of 25 μL of MTTsolution (5 mg/mL in PBS) was added to each well, and after 2 h ofincubation at 37° C., 100 μL of the extraction buffer (20% w/v of SDS,50% N,N-dimethyl formamide, pH 4.7) were added. After an overnightincubation at 37° C., the optical density at 600 rim was measured usingan ELISA reader.

Molecular Modeling.

Molecular modeling of AHNP has been performed as described previously.Other AHNP analogues were designed by comparative modeling using AHNP asa template. To that end, point mutations or deletions have beenintroduced in the AHINP sequence using the “Protein Design” applicationof program QUANTA (Molecular Simulation, MA). Each analogue has beenevaluated for the backbone and side chain orientation and solventeffects using a combination of energy minimization (CHARMM) andmolecular dynamics simulations at room temperature (300′K) and at 600′K.Low energy conformers were further minimized and compared with AHNP andthe native conformation of the template CDR3 loop of rhumAb 4D5.

Radiolabeling and Chemistry

For the radiolabeling of the AHNP with ^(99m)Tc, diethylenetriaminepentaacetate (DTPA) was used as the chelating agent. Since thecomplexation constant of ^(99m)Tc-DTPA is moderate,6-Hydrazinopyridine-3-carboxylic acid (HYNIC) and tricine can be usedfor ^(99m)Tc complexation.

HYNIC synthesis: the preparation of 6-hydrazinopyridine-3-carboxylicacid (HYNIC) is going to be carried out in a similar methods reported byAbrams et. al. [74].

Conjugation of HYNIC to AHNP: The conjugation of HYNIC to AHNP wasperformed in a conventional way. Briefly, To a solution of AHNP (1 mgAHNP dissolved in 1 mL 0.1 M pH 8.0 HEPES buffer) was added a fresh 20mg/mL solution of SHNH in dry

DMF dropwise with agitation (5:1 molar ratio of SHNH/peptide). Thevolume of DMF added was less than 10% of the total volume. The reactionmixture was incubated at room temperature for 30-60 min, and thenpurified by semi-preparative RP HPLC. The collection was dried by rotaryevaporation and lyophilized. The molecular weight of the coupled peptidewas determined by ESI-MS.

^(99m)Tc labeling of AHNP: 10 μL (about 1 μg) of conjugated peptide inwater, 50 μL pH 5.2 0.25 M ammonium acetate, 10 to about 100 μL (1-10mCi) of ^(99m)Tc-pertechnetate, 15 μL of a 100 μg/μL Tricine watersolution will be mixed together. To the mixture will be added 4-8 μgSnCl₂ in 10 μL 0.01 N HCl. After incubation at room temperature for30-60 min, the labeled peptide will be analyzed by reversed-phase (RP)HPLC, with water/acetonitrile containing 0.1% TFA as mobile phase, andpurified by size-exclusion (SE) HPLC with 0.1 M pH 7.2 phosphate bufferas mobile phase and fractionating. The fraction with the highestradioactivity would be used for in vitro and in vivo tests.

DTPA Chelation

Briefly, 1 mg of AHNP in 1 mL pH 8.0 to about 8.5 0.25 M bicarbonatebuffer, a suspension of the cyclic DTPA anhydride in 50 μL DMF will bemixed with agitation. The final DTPA/peptide molar ratio was about 5 toabout 10:1. After 30 min incubation at room temperature, the coupledpeptide will be purified by semi-preparative RP HPLC withwater/acetonitrile containing 0.1% TFA as mobile phase. The collectionwas dried by rotary evaporation and lyophilyzation. The molecular weightof the coupled peptide was determined by ESI-MS.

^(99m)Tc labeling: The DTPA-conjugated peptide was labeled with ^(99m)Tcin neutral medium. 1 to about 5 μg (10 to about 50 μL) of the DTPA-HNERmsolution was mixed with 10 μL (1 to about 10 mCi) of pertechnetae and 10μL 4 to about 8 μg SnCl₂ in 0.01 N HCl. After incubation at roomtemperature for 30 min, the radiolabel was analyzed by RP HPLC withwater/acetonitrile containing 0.1% TFA as mobile phase, and purified bySE HPLC with 0.1 M pH 7.2 phosphate buffer as mobile phase andfractionating. The fraction with highest radioactivity was used for invitro and in vivo tests.

DOTA Chelation

DOTA is commercial available. It was activated by NHS and ECD first andthen coupled to AHNP. The procedure of coupling is based on the work ofLewis et al. [75]. Briefly, Activated DOTA was prepared (4° C., 45 min)using DOTA, sodium bicarbonate, sulfo-NHS, and EDC at 10:30:10:1 molarratios. Conjugation was carried out by adding a 30-90-fold molar excessof activated DOTA to AHNP, adjusting pH to 8, and incubating forovernight h at 4° C. Purification was achieved by RP HPLC withwater/acetonitrile containing 0.1% TFA as mobile phase. The collectionwas dried by rotary evaporation and lyophilysation. The molecular weightof the conjugate was determined by ESI-MS.

^(99m)Tc labeling was performed as described above for DOTA chelation.

Blood Clearance.

AHNP labeled with ^(99m)Tc was used to estimate the blood clearance innude mice. Each animal of four groups received 5 μCi of theradioactivity through i.v. injection. After a given time post injection,the animals were anesthesized and their blood was collected viaretro-orbital sinus. The radioactivity in the blood samples, expressedas percentage injection dose per gram of the blood sample (% i.d./g,n=3+SE), was plotted against the time intervals between the injection ofthe radioactivity and the collection of the blood samples.

Animal Tumor Imaging and Biodistribution of AHNP.

NCr homozygous athymic (nude) mice were purchased from the NationalCancer Institute (Bethesda, Md.). An aliquot of 2×10⁶ T6-17 tumor cells(NIH 3T3 cells stably transfected with p185^(HER2/neu)) were suspendedin 200 ul of PBS and injected subdermally. Six days after tumorxenograft, tumors reached about 200-230 mm³ in volume. Mice bearingT6-17 xenografted tumors received purified ^(99m)Tc labeled AHNP. Theimaging was taken 30 minutes and 90 minutes postinjection andbiodistributions were carried out after the imaging was finished (Table2).

Combination Treatment.

Both MCF-7 parental strain (MCF/Par) and HER-2-overexpressing (MCF-7/H2)tumor-cells were treated with tamoxifen alone (TAM); with ANHP analogFCGDGFYACYMDV (SEQ ID NO: 143) alone (1 μg/ml); with AHNP analogYCDGFYACYMDV (SEQ ID NO: 113) alone (1 μg/ml); with tamoxifen and theformer (SEQ ID NO: 143); and with tamoxifen and the latter (SEQ ID NO:113) (FIG. 9).

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TABLE 1 Summary of Biophysical Properties and Biological Activities forAHNP Analogs AHNP binding to HER2 analogs Composition ^(k)on × 10² M⁻¹s⁻¹ ^(k)off × 10⁻⁴ s⁻¹ K_(D) μM % inhibition^(a) solubulity^(b) 1FCDGFYACYMDV-NH2 14.10 4.53 0.32 35.5 limited 2 FCDGFYACYMDV-OH 9.906.43 0.65 22.3 limited 3 YCDGFYACYMDV-NH2 19.60 2.94 0.15 59.6 limited 4dFCDGFYACdYMDV-NH2 17.20 6.03 0.35 26.0 good 5 FCDGFYACYMDVK-NH2 6.175.12 0.83 20.0 limited 6 FCDGFYACYKDV-OH 2.19 12.50 5.70 8.4 good 7GFCDGFYACYMDV-OH 11.10 8.08 0.73 15.8 good 8 FCGDGFYACYMDV-OH 9.22 7.010.76 18.4 good 9 FCDGFYACYMDV-OH 2.98 10.60 3.56 12.8 good^(a)Inhibition of T6-17 cell proliferation in MTT assays. Each valuerepresents an average of at least four samples. Standard error did notexceed 535 for any of the studied analogues. ^(b)Peptide solubility isindicated as good if the peptide can be readily dissolved in the PBSbuffer at 1 mg/mL, and as limited if pH adjustment is required fordissolving the peptide.

TABLE 2 Biodistribution of the ^(99m)Tc labeled AHNP ^(99m)Tc-AHNPOrgans Standard N = 3 ID %/g Deviation Liver 1.73 0.55 Heart 0.18 0.038Kidney 8.6 3.0 Lung 0.39 0.12 Spleen 0.92 0.17 Stomach 1.8 0.25 SmallIntestine 0.30 0.06 Large Intestine 0.46 0.29 Muscle 0.083 0.032 Tumor0.24 0.050 Blood 0.59 0.15 Tumor/Muscle 3.0 0.64 Tumor/Blood 0.43 0.15

1. An isolated cyclic peptide having an amino acid sequence selectedfrom the group consisting essentially of: YCDGFYACYMDV-NH₂ (SEQ ID NO:113 wherein the carboxy terminus is amidated); FCDGFYACYMDVK-NH₂ (SEQ IDNO: 123 wherein the carboxy terminus is amidated); and GFCDGFYACYMDV-OH(SEQ ID NO: 134 wherein the carboxy terminus is hydroxylated).
 2. Apharmaceutical composition comprising a peptide according to claim 1,and a pharmaceutically acceptable carrier or diluent.
 3. The compositionof claim 2, wherein the peptide is conjugated to a detectible agentand/or a cytotoxic agent.
 4. The composition of claim 3, wherein thecytotoxic agent is a radioisotope.
 5. The composition of claim 4,wherein the radioisotope is ^(99m)Tc, ⁹⁰Y, or ¹⁸F.